HRP20040242A2 - Protein knobs - Google Patents
Protein knobs Download PDFInfo
- Publication number
- HRP20040242A2 HRP20040242A2 HR20040242A HRP20040242A HRP20040242A2 HR P20040242 A2 HRP20040242 A2 HR P20040242A2 HR 20040242 A HR20040242 A HR 20040242A HR P20040242 A HRP20040242 A HR P20040242A HR P20040242 A2 HRP20040242 A2 HR P20040242A2
- Authority
- HR
- Croatia
- Prior art keywords
- protein
- subunit
- cysteine
- hcg
- knot
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Endocrinology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
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Description
Vladin interes Government interest
Ovaj rad je podržan od strane Nacionalnog Instituta za Zdravstvena Odobrenja NICHD HD14907 i NICHD HD38547. Ovaj izum je učinjen uz podršku Vlade Sjedinjenih Američkih Država. Vlada može posjedovati određena prava u predloženom izumu. This work was supported by National Institutes of Health Grants NICHD HD14907 and NICHD HD38547. This invention was made with the support of the United States Government. The government may own certain rights in the proposed invention.
Područje izuma Field of invention
Predloženi izum se odnosi na područje proteinskog označavanja. The proposed invention relates to the field of protein labeling.
Uvod Introduction
Metode označavanja proteina za upotrebu u analizi interakcija protein-protein ili u proteinskoj purifikaciji trenutačno obuhvaćaju fuziju oznake za karboksi- ili amino- terminalne krajeve proteina, ili umetanje ostataka u petlju proteina. Svaka od ovih metoda ima svoja ograničenja. Na primjer, može biti poželjno označiti protein, bilo sa obojenom molekulom ili proteinom, na specifičnim lokacijama na proteinu, radije nego biti ograničen na pričvršćivanje molekula na proteinske krajeve. Ovo uključuje studije dizajnirane da testiraju interakcije proteina sa makromolekulama, kao u slučaju hCG-a i njegovog receptora. Dodatno, u slučajevima gdje su terminalni krajevi proteina uključeni u proteinsku funkciju, označavanje proteinskih krajeva može biti nepoželjno. Uz poštovanje umetanju oznake u petlju proteina, ova metoda je ograničena zato što veličina oznake obično mora biti relativno mala (nekoliko ostataka), osim ako je oznaka umetnuta između proteinskih domena. Postoje situacije kada bi bilo poželjno pričvrstiti testere različitih veličina na površinu proteina. Nadalje, modifikacije koje uključuju uporabu cisteinskog ostatka su također komplicirane jer protein može sadržavati druge cisteinske ostatke koji će zahtijevati protekciju, ili cistein može biti blokiran i proteinska denaturacija se može pojaviti pri uklanjanju blokirajućeg ostatka. Kao rezultat, označeni proteini producirani ovim metodama imaju ograničenu upotrebu i aplikaciju u industriji. Methods of labeling proteins for use in the analysis of protein-protein interactions or in protein purification currently involve fusion of tags to the carboxy- or amino-terminal ends of proteins, or insertion of residues into protein loops. Each of these methods has its limitations. For example, it may be desirable to label a protein, either with a dye molecule or a protein, at specific locations on the protein, rather than being limited to attaching molecules to protein ends. This includes studies designed to test protein interactions with macromolecules, as in the case of hCG and its receptor. Additionally, in cases where the terminal ends of the protein are involved in protein function, labeling the protein ends may be undesirable. With respect to the insertion of a tag into a protein loop, this method is limited because the size of the tag must usually be relatively small (a few residues), unless the tag is inserted between protein domains. There are situations when it would be desirable to attach probes of different sizes to the protein surface. Furthermore, modifications involving the use of a cysteine residue are also complicated because the protein may contain other cysteine residues that will require protection, or the cysteine may be blocked and protein denaturation may occur upon removal of the blocking residue. As a result, labeled proteins produced by these methods have limited use and application in industry.
Na primjer, napori da se identificiraju porcije humanog korionskog gonadotropina (hCG) koje kontaktiraju lutropinski receptor su bili onemogućeni kompleksnom strukturom hormona i vjerojatnošću da imterreagira sa receptorom na multiplim lokacijama. Kristalna struktura humanog korionskog gonadotropina (hCG) je otkrila da vlakno njegove β-podjedinice okružuje α-podjedinicu poput sigurnosnog pojasa (Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J. & Isaacs, N. W. (1994) Nature 369, 455-461; Wu, H., Lustbader, J. W., Liu, Y., Canfield, R. E. & Hendrickson, W. A. (1994) Structure 2, 545-558.). Za razliku od mnogih dimernih proteina koji su stabilizirani u potpunosti sa interpodjediničnim kontaktima, čini se da je hCG osiguran većinom sa svojim sigurnosnim pojasom; otkriveno je da eliminacija disulfida što «kvači» karboksiterminalni kraj sigurnosnog pojasa za Cys26 u jezgri β-podjedinice uzrokuje disrupciju sekrecije hCG-a (Suganuma, N., Matzuk, M. M.& Boime, I: (1989) J. Biol. Chem. 264, 19302-19307), vjerojatno destabilizacijom heterodimera. Evolucijske prednosti ovog neobičnog strukturnog rasporeda ostaju nepoznate, i mogu se odnositi na otkriće da dozvoljava kretnje podjedinica unutar heterodimera, fenomen koji je detektiran za vrijeme vezanja nekih hCG analoga za FSH receptore (Wang, Y. H., Bernard, M. P. & Moyle, W. R. (2000) Mol. Cell. Endocrinol. 170, 67-77). For example, efforts to identify the portions of human chorionic gonadotropin (hCG) that contact the lutropin receptor have been hampered by the hormone's complex structure and the likelihood that it interacts with the receptor at multiple sites. The crystal structure of human chorionic gonadotropin (hCG) revealed that its β-subunit fiber surrounds the α-subunit like a seat belt (Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan , F. J. & Isaacs, N. W. (1994) Nature 369, 455-461; Wu, H., Lustbader, J. W., Liu, Y., Canfield, R. E. & Hendrickson, W. A. (1994) Structure 2, 545-558. Unlike many dimeric proteins that are stabilized entirely by intersubunit contacts, hCG appears to be secured mostly by its safety belt; elimination of the disulfide that "clutches" the carboxy-terminal end of the safety belt for Cys26 in the β-subunit core has been found to cause disruption of hCG secretion (Suganuma, N., Matzuk, M. M. & Boime, I: (1989) J. Biol. Chem. 264 , 19302-19307), probably by destabilizing the heterodimer. The evolutionary advantages of this unusual structural arrangement remain unknown, and may relate to the discovery that it allows movement of subunits within the heterodimer, a phenomenon that has been detected during the binding of some hCG analogs to FSH receptors (Wang, Y. H., Bernard, M. P. & Moyle, W. R. (2000) Mol. Cell. Endocrinol. 170, 67-77).
Receptori sva tri razreda glikoproteinskih hormona, uključujući hCG, su spareni u G-proteine i imaju velike ekstracelularne domene koje sadrže multipla leucinom-bogata ponavljanja (Segaloff, D.L. & Ascolli, M. (1993) Endocr. Rev. 14, 324-347). Kasnija otkrića sugeriraju da bi ekstracelularna domena mogla biti oblikovana poput potkove, slično dijelovima drugih proteina s leucinom-bogatim ponavljanjima (Kobe, B. & Deisenhofer, J. (1993) Nature 366, 751-756). Čini se da dvije regije ekstracelularne domene pridonose afinitetu i specifičnosti kod vezanja liganda. Afinitet hCG-a prema naizmjenično spojenim i okrnjenim LHR analozima je sličan onome prema intaktnom receptoru, što sugerira da ostaci na aminoterminalne dvije trećine ekstracelularne domene formiraju mjesto visokog afiniteta za vezanje liganda (Braun, T., Schofield, P. R. & Sprengel, R. (1991) EMBO.J. 10, 1885-1890; Thomas, D., Rozell, T. G., Liu, X. & Segaloff, D. L. (1996) Mol. Endocrinol. 10, 760-768). Ostaci na karboksiterminalnoj petini humane LHR ekstacelularne domene interferiraju sa vezanjem ne-humanih lutropina sisavaca, otkriće je koje ukazuje na to da su kontakti između ovog dijela hormona i receptora primarno sterični u prirodi (Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem. J. 335, 611-617). Receptors for all three classes of glycoprotein hormones, including hCG, are G-protein coupled and have large extracellular domains containing multiple leucine-rich repeats (Segaloff, D.L. & Ascolli, M. (1993) Endocr. Rev. 14, 324-347) . Later discoveries suggested that the extracellular domain might be shaped like a horseshoe, similar to parts of other proteins with leucine-rich repeats (Kobe, B. & Deisenhofer, J. (1993) Nature 366, 751-756). Two regions of the extracellular domain appear to contribute to ligand binding affinity and specificity. The affinity of hCG for alternately spliced and truncated LHR analogs is similar to that for the intact receptor, suggesting that residues in the amino-terminal two-thirds of the extracellular domain form a high-affinity ligand-binding site (Braun, T., Schofield, P. R. & Sprengel, R. ( 1991) EMBO.J. 10, 1885-1890; Thomas, D., Rozell, T.G., Liu, X. & Segaloff, D.L. (1996) Mol. Endocrinol. 10, 760-768). Residues in the carboxy-terminal fifth of the human LHR extracellular domain interfere with the binding of non-human mammalian lutropins, a finding that suggests that the contacts between this part of the hormone and the receptor are primarily steric in nature (Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) ) Biochem. J. 335, 611-617).
Površine hCG, hFSH, te hTSH koje će najvjerojatnije kontaktirati s LHR, FSHR, te TSHR ostaju upitne. Nađeno je da karboksiterminalni kraj α-podjedinice, koji graniči sa porcijom sigurnosnog pojasa u heterodimera (Wu, H., Lustbader, J. W., Liu, Y., Canfield, R. E. & Hendrickson, W. A. (1994) Structure 2, 545-558), utječe na afinitet svih glikoproteinskih hormona prema njihovim receptorima (Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J. & Isaacs, N. W: (1994) Nature 369, 455-461; Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem.J. 335, 611-617) i bio je predložen kao receptorski kontakt prije više od 25 godina (Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J.W., Canfield, R. E., Machin, K. J., Morgan, F. J. & Isaacs, N. W: (1994) Nature 369, 455-461). Zajedno s podacima o strukturi i funkciji hormona, ove opservacije dovele su nas do iznimno različitih pogleda na kompleks hormon-receptor (Moyle, W. R., Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031), počevši od onog gdje hormon kontaktira sa konkavnom površinom ekstracelularne domene receptora (Jiang, X., Dreano, M., Buckler, D. R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W. A., Tayar, N. E. & el Tayar, N. (1995) Structure 3, 1341-1353), do onog gdje kotaktira s njegovim rubom (Moyle, W. R., Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031). Sva gledišta na hCG-LHR kompleks sugeriraju da su porcije α-podjedinične petlje 2 okrenute ka receptoru, ali još se mora determinirati da li ova porcija hormona participira u kontaktima s receptorom. Izviješteno je da mutacija ove petlje reducira aktivnost hCG-a, (Peng, K. C., Bousfield, G. R., Puett, D. & Ward, D. N. (1996) Journal of Protein Chemistry 15, 547-552; Xia, H., Chen, F. & Puett, D. (1994) Endocrinol. 134, 1768-1770) što navodi na to da možda stvara esencijalne kontakte s LHR. The surfaces of hCG, hFSH, and hTSH that are most likely to contact LHR, FSHR, and TSHR remain questionable. The carboxy-terminal end of the α-subunit, which borders the seat belt portion of the heterodimer (Wu, H., Lustbader, J. W., Liu, Y., Canfield, R. E. & Hendrickson, W. A. (1994) Structure 2, 545-558), was found to affects the affinity of all glycoprotein hormones for their receptors (Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J. & Isaacs, N. W: (1994) Nature 369 , 455-461; Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem.J. 335, 611-617) and was proposed as a receptor contact more than 25 years ago (Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J. & Isaacs, N.W.: (1994) Nature 369, 455-461). Together with data on hormone structure and function, these observations have led us to very different views of the hormone-receptor complex (Moyle, W. R., Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y. & Wang , Y. (1995) J. Biol. Chem. 270, 20020-20031), beginning where the hormone contacts the concave surface of the extracellular domain of the receptor (Jiang, X., Dreano, M., Buckler, D. R., Cheng, S. , Ythier, A., Wu, H., Hendrickson, W. A., Tayar, N. E. & el Tayar, N. (1995) Structure 3, 1341-1353), to where it contacts its edge (Moyle, W. R., Campbell, R. K. , Rao, S.N.V., Ayad, N.G., Bernard, M.P., Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031). All views on the hCG-LHR complex suggest that portions of the α-subunit loop 2 face the receptor, but it remains to be determined whether this portion of the hormone participates in receptor contacts. Mutation of this loop has been reported to reduce hCG activity, (Peng, K. C., Bousfield, G. R., Puett, D. & Ward, D. N. (1996) Journal of Protein Chemistry 15, 547-552; Xia, H., Chen, F & Puett, D. (1994) Endocrinol. 134, 1768-1770) suggesting that it may make essential contacts with the LHR.
Interakcije protein-receptor su ključ u razumijevanju funkcije i regulacije staničnog ponašanja. Dobro razumijevanje interakcija protein-receptor je neophodno za mnoge nove pokušaje, kao što je dizajniranje novih farmaceutskih lijekova. Ipak, trenutačno postoje mnoge limitacije u shvaćanju interakcija protein-receptor. Korisno je razumijeti i strukturu proteinske konformacije i proteinske funkcije, kao i način na koji struktura proteina interreagira s receptorom (koji je također obično protein). Preko znanja i iskustva, jedan aspekt proteina može ponekad biti ekstrapoliran ako su drugi aspekti poznati. Interakcije protein-receptor mogu biti modelirane kompjuterski, ali modeliranje je složen zadatak, posebno zato što su molekule fleksibilne i poprimaju više konformacija koje su slične energije. Modeliranje procesa protein-receptorskog vezanja je također komplicirano zbog karakteristika receptora, liganda, te solventnih potreba koje se trebaju razmotriti. Protein-receptor interactions are the key to understanding the function and regulation of cellular behavior. A good understanding of protein-receptor interactions is essential for many new endeavors, such as designing new pharmaceutical drugs. However, there are currently many limitations in understanding protein-receptor interactions. It is useful to understand both the structure of protein conformation and protein function, as well as how the protein structure interacts with the receptor (which is also usually a protein). Through knowledge and experience, one aspect of a protein can sometimes be extrapolated if other aspects are known. Protein-receptor interactions can be modeled computationally, but modeling is a complex task, especially since molecules are flexible and assume multiple conformations of similar energy. Modeling the protein-receptor binding process is also complicated by the characteristics of the receptor, ligand, and solvent requirements that must be considered.
Kao dodatak modeliranju, još jedan pristup razumijevanju interakcija protein-receptor jest stvaranje promjena u strukturi proteina, strukturi receptora, ili u uvjetima pod kojima stupaju u interakciju u eksperimentalnoj okolini, te mjerenje efekta promjene na vezanje, te prema tome, i funkcije proteina ili manjak toga. Ove eksperimentalne tehnike također imaju svoja ograničenja zbog teškoće pri manipuliranju proteinom ili receptorom, te ograničenja zbog mogućnosti mjerenja samih promjena. Upravo zato, izum koji bi mogao poboljšati točnost eksperimentalnih podataka dosljedno uzrokujući protein-receptorsku interakciju ili dosljedno inhibirajući protein-receptorsku interakciju, bi bilo poboljšanje u struci zato što bi omogućilo determinaciju funkcije ili manjak funkcije koji je pri tom izazvan. In addition to modeling, another approach to understanding protein-receptor interactions is to create changes in protein structure, receptor structure, or in the conditions under which they interact in an experimental environment, and measure the effect of the change on binding, and therefore protein function or deficiency. toga. These experimental techniques also have their limitations due to the difficulty in manipulating the protein or receptor, and limitations due to the ability to measure the changes themselves. Therefore, an invention that could improve the accuracy of experimental data by consistently causing protein-receptor interaction or consistently inhibiting protein-receptor interaction would be an improvement in the art because it would enable determination of the function or lack of function induced thereby.
U principu, identifikacija porcija hormona i receptora koji međusobno kontaktiraju bi trebala biti lako objašnjena upotrebljavajući na mjesto-usmjerenu mutagenezu. Na nesreću, mutacije karboksiterminala α-podjedinice i sigurnosnog pojasa β-podjedinice mijenjaju pozicije podjedinica unutar heterodimera (Jiang, X., Dreano, M., Buckler, D. R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W. A., Tayar, N. E. & el Tayar, N. (1995) Structure 3, 1341-1353; Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495), te su na taj način otežale interpretaciju utjecaja ovih ključnih dijelova hormona na njegovu funkciju. Dalje, mutacije koje mijenjaju aktivnost hCG-a mogu to činiti mijenjanjem ključnih kontakta LHR, zamijenjivanjem pozicija njegovih podjedinica, ili oboje. Uistinu, mutacije karboksiterminala α-podjedinice (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495, Chen, F., Wang, Y. & Puett, D. (1992) Mol. Endocrinol. 6, 914-919) i sigurnosnog pojasa (Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764; Campbell, R. K., Bergert, E. R., Wang, Y., Morris, J. C. & Moyle, W. R. (1997) Nature Biotech. 15, 439-443; Grossman, M., Szkudlinski, M. W., Wong, R., Dias, J. A., Ji, T. H. & Weintraub, B. D. (1997) J. Biol. Chem. 272, 15532-15540; Lindau-Shepard, B., Roth, K. E. & Dias, J. A. (1994) Endocrinol. 135, 1235-1240), koje imaju jaki utjecaj na hormon-receptorske interakcije, također mijenjaju pozicije podjedinica unutar heterodimera (Wang, Y. H., Bernard, M. P. & Moyle, W. R. (2000) Mol. Cell. Endocrinol. 170, 67-77; Moyle, W. R., Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031). Alternativna metoda za identifikaciju koje porcije proteina i receptora međusobno reagiraju uključuje identifikaciju ostataka koji ne kontaktiraju receptor. Ovi ostaci se mogu identificirati s puno većom sigurnošću. Međutim, bilo je jako teško pribaviti testere za upotrebu u identifikaciji ovih ostataka. Prema tome, neophodne su poboljšane metode za proizvodnju testera za upotrebu u analizi proteinskih interakcija. In principle, the identification of portions of hormones and receptors that contact each other should be easily explained using site-directed mutagenesis. Unfortunately, mutations in the α-subunit carboxy-terminal and β-subunit seat belts alter the positions of the subunits within the heterodimer (Jiang, X., Dreano, M., Buckler, D. R., Cheng, S., Ythier, A., Wu, H., Hendrickson , W. A., Tayar, N. E. & el Tayar, N. (1995) Structure 3, 1341-1353; Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495), and thus made it difficult to interpret the influence of these key parts of the hormone on its function. Further, mutations that alter hCG activity may do so by altering key LHR contacts, swapping the positions of its subunits, or both. Indeed, mutations in the carboxy-terminal α-subunit (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495, Chen, F., Wang, Y. & Puett, D. (1992) Mol. Endocrinol. . 6, 914-919) and seat belt (Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764; Campbell, R. K., Bergert, E. R. , Wang, Y., Morris, J.C. & Moyle, W.R. (1997) Nature Biotech. 15, 439-443; Grossman, M., Szkudlinski, M.W., Wong, R., Dias, J.A., Ji, T.H. & Weintraub, B.D. (1997) J. Biol. Chem. 272, 15532-15540; Lindau-Shepard, B., Roth, K. E. & Dias, J. A. (1994) Endocrinol. 135, 1235-1240), which have a strong influence on hormone-receptor interactions , also change subunit positions within the heterodimer (Wang, Y. H., Bernard, M. P. & Moyle, W. R. (2000) Mol. Cell. Endocrinol. 170, 67-77; Moyle, W. R., Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M.P., Han, Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031). An alternative method for identifying which portions of the protein and receptor interact involves identifying residues that do not contact the receptor. These remains can be identified with much greater certainty. However, it has been very difficult to obtain testers for use in the identification of these remains. Therefore, improved methods for producing probes for use in protein interaction analysis are needed.
Kao dodatak upotrebi u analizi proteinskih interakcija, testeri ili proteini označeni na specifičnom mjestu na molekuli osiguravaju neprocjenjiv alat za istraživanja. Međutim, kao što je prethodno opisano, suvremene metode uključuju označavanje ili pričvršćivanje testera za terminalne krajeve molekule, ograničavajući njihovu upotrebu. Ostale metode uključuju komplicirane procedure koje zahtijevaju različite reakcije i protektivne skupine za produkciju modifikacija na specifičnom mjestu, bez modificiranja ostalih amino kiselina i strukture funkcionalnog proteina. Prema tome, poželjne su poboljšane metode za označavanje specifičnog mjesta. In addition to their use in the analysis of protein interactions, probes or proteins tagged at a specific site on a molecule provide an invaluable research tool. However, as previously described, current methods involve labeling or attaching the probes to the terminal ends of the molecule, limiting their use. Other methods involve complicated procedures that require different reactions and protective groups to produce site-specific modifications without modifying other amino acids and the structure of the functional protein. Therefore, improved methods for site-specific labeling are desirable.
Proteinska purifikacija je često neophodan, iako ponekad tegoban proces. Purificirani proteini mogu biti potreban intermedijarni produkt znanstvenog eksperimenta, ili mogu biti krajnji produkt. Proteinska purificiranost je često presudna za eksperimentalnu i terapeutsku uspješnost. U nekim slučajevima, gdje su proteini za injiciranje pomno ispitani od strane Administracije za Hranu i Lijekove, bilo kakvi kontaminati se moraju ukloniti ili se mora dokazati da su bezopasni. Kao dodatak purificiranosti, proteini moraju zadržati svoju biološku aktivnost. Protein purification is often a necessary, if sometimes difficult, process. Purified proteins may be a necessary intermediate product of a scientific experiment, or they may be the final product. Protein purity is often crucial for experimental and therapeutic success. In some cases, where injectable proteins have been scrutinized by the Food and Drug Administration, any contaminants must be removed or proven harmless. In addition to being purified, proteins must retain their biological activity.
Postoji niz različitih metoda za purifikaciju proteina koje su trenutno poznate u struci, iako sve imaju neka ograničenja. Kromatografija koja vrši ekskluziju po veličini pomaže pri grubim separacijama, ali to nije precizna metoda i zahtijeva da uzorci budu koncentrirani. Gel elektroforeza dozvoljava precizniju separaciju proteina u mješavini, ali je praktična samo za male uzorke. Afinitetna kromatografija je korisna metoda, ali tipično zahtijeva da su neki od iniciijalnih kontaminata već filtrirani. Prema tome, izum koji bi mogao pomoći u preciznoj separaciji proteina u nekoliko koraka bilo bi signifikantno poboljšanje u struci, osobito ako bi izum mogao biti povećan u određenom razmjeru tako da purificira veće količine, a bez žrtvovanja kvalitete purficiranosti. There are a number of different methods for protein purification currently known in the art, although they all have some limitations. Size exclusion chromatography helps with coarse separations, but it is not a precise method and requires the samples to be concentrated. Gel electrophoresis allows a more precise separation of proteins in a mixture, but is only practical for small samples. Affinity chromatography is a useful method, but typically requires that some of the initial contaminants have already been filtered out. Therefore, an invention that could assist in the precise separation of proteins in several steps would be a significant improvement in the art, especially if the invention could be scaled up to purify larger quantities without sacrificing the quality of purification.
Kao dodatak ovim upotrebama, čvorovi se mogu koristiti da 'prekriju' specifične površine na proteinima. Ova vrsta upotrebe se aplicira u dizajniranju prolijekova koji se mogu upotrijebiti za ciljanje tumora ili drugih nepoželjnih tkiva, kao što su ona pronađena u jajnicima neplodnih žena s dijagnozom sindroma policističnih jajnika. Na taj način, pričvršćivanje čvora u blizini aktivnog mjesta toksina ili toksičnog enzima bi dozvolilo upotrebu toksičnog enzima u pacijenata. Jednom kad toksin ili enzim dospiju u tkivo koje sadrži enzim sposoban za cijepanje karike koja je pričvrstila čvor, toksično ili enzimatsko djelovanje može biti ponovo uspostavljeno. Očekuje se da ova strategija smanji prisutnost nepoželjnih nuspojava koje inače mogu ograničiti upotrebljivost toksina ili enzima. Na isti način, čvorovi bi se mogli iskoristiti za blokiranje aktivnosti posrednika kao što su PTEN koji potiču apoptozu. In addition to these uses, knots can be used to 'cover' specific surfaces on proteins. This type of use is applied in the design of prodrugs that can be used to target tumors or other unwanted tissues, such as those found in the ovaries of infertile women diagnosed with polycystic ovary syndrome. Thus, attaching the node near the active site of the toxin or toxic enzyme would allow the use of the toxic enzyme in patients. Once the toxin or enzyme reaches a tissue that contains an enzyme capable of cleaving the link that attached the knot, the toxic or enzymatic action can be re-established. This strategy is expected to reduce the presence of undesirable side effects that may otherwise limit the utility of the toxin or enzyme. In the same way, nodes could be used to block the activity of mediators such as PTEN that promote apoptosis.
Sažetak izuma Summary of the invention
Predloženi izum se odnosi na kompozicije koje se sastoje od proteina označenih na specifičnom mjestu sa čvorom te metoda za stvaranje i korištenje ovih kompozicija. Čvor se odnosi na oznaku za protein koja može biti kustomizirana za specifičnu upotrebu. The proposed invention relates to compositions consisting of proteins marked at a specific site with a knot and methods for creating and using these compositions. A node refers to a tag for a protein that can be customized for a specific use.
Iz jednog aspekta izuma, proteini označeni s čvorom na specifičnom mjestu proteina su obznanjeni. Protein označen na specifičnom mjestu sadrži čvor, porciju repa, te proteinsku porciju. Čvor je se sastoji od oznake ili testera aspekta proteina i ima cisteinski ostatak. Porcija repa je locirana između porcije čvora i proteinske porcije. Proteinska porcija ima cisteinski ostatak supstituiran umjesto nativne aminokiseline na željenom mjestu označavanja. Cisteinski ostaci čvora i proteinske porcije formiraju disulfidnu vezu. Iz dodatnog aspekta izuma, porcija repa može sadržavati proteazu ili drugo mjesto za cijepanje. In one aspect of the invention, knot-tagged proteins at a specific protein site are disclosed. A protein labeled at a specific location contains a knot, a tail portion, and a protein portion. The knot consists of a tag or probe aspect of the protein and has a cysteine residue. The tail portion is located between the knot portion and the protein portion. The protein portion has a cysteine residue substituted for the native amino acid at the desired labeling site. The cysteine residues of the knot and the protein portions form a disulfide bond. In an additional aspect of the invention, the tail portion may contain a protease or other cleavage site.
Iz drugog aspekta izuma, metode za proizvodnju proteina označenih s čvorom na specifičnom mjestu su obznanjene. Ove metode uključuju selekcioniranje željenog proteina za označavanje, lociranje specifičnog mjesta na željenom proteinu za označavanje, proizvodnju željenog proteina, u kojem je cisteinski ostatak supstituiran na specifičnom mjestu za označavanje u željenom proteinu, u kojem se željeni protein nadalje sastoji od porcije repa na jednom kraju proteina i čvora na kraju proteinske porcije, u kojem čvor sadrži cisteinski ostatak, i gdje cisteinski ostatak čvora formira disulfidnu vezu s cisteinskim ostatkom u proteinskoj porciji. Odnoseći se na ovaj aspekt izuma, metode za pričvršćivanje čvorova za hCG na specifičnim mjestima su obznanjene. Ove metode uključuju umetanje konstrukcija sposobnih za ekspresiju nativnog hCGβ ili hCGβ-S138C, i nativnog hCGα ili hCGα-cistein supstituiranih analoga u stanicu za ko-ekspresiju, te fuziju čvora za ostatak 140 ili 145 hCGβ. In another aspect of the invention, methods for producing site-specific knot-tagged proteins are disclosed. These methods include selecting a desired tagging protein, locating a specific site on the desired tagging protein, producing the desired protein, wherein a cysteine residue is substituted at the specific tagging site in the desired protein, wherein the desired protein further comprises a tail portion at one end of the protein and a knot at the end of the protein portion, in which the knot contains a cysteine residue, and where the cysteine residue of the knot forms a disulfide bond with a cysteine residue in the protein portion. In relation to this aspect of the invention, methods for attaching knots to hCG at specific locations are disclosed. These methods include inserting constructs capable of expressing native hCGβ or hCGβ-S138C, and native hCGα or hCGα-cysteine substituted analogs into a cell for co-expression, and junctional fusion to residue 140 or 145 of hCGβ.
Iz slijedećeg aspekta izuma, metode proteinske purifikacije koje upotrebljavaju proteine označene na specifičnom mjestu u ovom izumu su obznanjene. Ove metode uključuju umetanje konstrukcije što enkodira protein u stanicu, pri čemu enkodirani protein sadrži cisteinski ostatak supstituiran na željenom mjestu za označavanje, porciju repa, koja ima cisteinski ostatak i mjesto cijepanja, na jednom kraju proteina, te porciju čvora na kraju porcije repa, osiguravajući uvjete koji dozvoljavaju ekspresiju konstrukcije, liziranje stanice, te purifikaciju proteina baziranu na karakteristikama čvora proteina. In a further aspect of the invention, methods of protein purification using site-tagged proteins are disclosed in the present invention. These methods involve inserting a protein-encoding construct into the cell, wherein the encoded protein contains a cysteine residue substituted at the desired labeling site, a tail portion, having a cysteine residue and a cleavage site, at one end of the protein, and a knot portion at the end of the tail portion, providing conditions that allow expression of the construct, cell lysis, and protein purification based on the characteristics of the protein knot.
Iz slijedećeg aspekta izuma, metode za korištenje po specifičnom mjestu modificiranih proteinskih čvorova su obznanjene. Proteinski čvorovi se mogu koristiti, na primjer, za mapiranje distanci među proteinima, testiranje površine protein-proteinskog interface-a, formiranje kompleksa između dva nesrodna proteina, testiranje strukture i funkcije protein čvor protein, za imobilizaciju proteina na površinama, za isporuku proteina stanicama, kao ciljani protein, i za proteinsku purifikaciju. Tko je iskusan u struci će prepoznati da protein označen na specifičnom mjestu sa kustomiziranom oznakom jest dragocjen alat za istraživanje s širokim dijapazonom upotrebe. In a further aspect of the invention, methods for using site-specifically modified protein knots are disclosed. Protein knots can be used, for example, to map distances between proteins, test the surface of a protein-protein interface, form a complex between two unrelated proteins, test the structure and function of a protein knot protein, to immobilize proteins on surfaces, to deliver proteins to cells, as a target protein, and for protein purification. Those skilled in the art will recognize that a site-specific protein labeled with a customized label is a valuable research tool with a wide range of uses.
Kratak opis crteža Brief description of the drawing
Slika 1: Trodimenzionalni prikaz hCG-a. Ilustracija ostataka α-podjedinice hCG-a koji mogu biti skenirani s karboksiterminalom β-podjedinice hCG-a. Glavne osi α- i β-podjedinice su prikazane tamnim i svijetlim sivim vrpcama, svaka ponaosob. β-rep je prikazan kao crna vrpca. Lokacije ugljika Cα cisteinskih supstitucija što su omogućile djelotvornu ukriženu vezu između ostatka α-podjedinice i cisteinskog testera su prikazane kao tamne kugle. Svijetlije sive kugle se odnose na ostatke što stvaraju manje količine ukriženih veza. Male blijede kugle odnose se na cisteinske supstitucije koje su dovele do beznačajne količine ukriženih veza. Obratiti pažnju na ostatke 90, 91 i 92 α-podjedinice, koji se čine odveć mobilni da bi se vidjeli u kristalnoj strukturi hCG-a, te proizvoljne pozicije ovih ostataka prikazane ovdje imaju namjeru samo naglasiti njihove očite sposobnosti da se zakvače za sigurnosni pojas. Figure 1: Three-dimensional representation of hCG. Illustration of residues of the hCG α-subunit that can be scanned with the carboxy-terminus of the hCG β-subunit. The principal axes of the α- and β-subunits are shown by dark and light gray bands, respectively. The β-tail is shown as a black band. The carbon locations of the Cα cysteine substitutions that enabled effective cross-linking between the α-subunit residue and the cysteine tester are shown as dark spheres. Lighter gray spheres refer to residues that form smaller amounts of cross-links. The small pale spheres refer to cysteine substitutions that led to an insignificant amount of cross-links. Note residues 90, 91, and 92 of the α-subunit, which appear too mobile to be seen in the crystal structure of hCG, and the arbitrary positions of these residues shown here are only intended to emphasize their apparent abilities to latch onto the seat belt.
Slika 2A: Vezanje hCG-a ili hCG analoga u kojih je α-podjedinični ostatak u petlji 2 supstituiran cisteinom. Figure 2A: Binding of hCG or hCG analogs in which the α-subunit residue in loop 2 is substituted with cysteine.
Slika 2B: cAMP akumulacija hCG-a ili hCG analoga u kojih je α-podjedinični ostatak u petlji 2 supstituiran cisteinom. Figure 2B: cAMP accumulation of hCG or hCG analogs in which the α-subunit residue in loop 2 is substituted with cysteine.
Slika 3A: Utjecaj cisteinskih supstitucija na α-podjediničnom karboksiterminalu. Vezanje hCG-a ili hCG analoga u kojih je α-podjedinični ostatak na karboksiterminalu supstituiran cisteinom. Figure 3A: Effect of cysteine substitutions on the α-subunit carboxyterminal. Binding of hCG or hCG analogs in which the α-subunit residue at the carboxy terminal is substituted with cysteine.
Slika 3B: Utjecaj cisteinskih supstitucija na α-podjediničnom karboksiterminalu. cAMP akumulacija hCG-a ili hCG analoga u kojih je α-podjedinični ostatak na karboksiterminalu supstituiran cisteinom. Figure 3B: Effect of cysteine substitutions on the α-subunit carboxyterminal. cAMP accumulation of hCG or hCG analogs in which the α-subunit residue at the carboxy terminal is substituted with cysteine.
Slika 4A: Vezanje hCG-a ili hCG analoga s β-repom pričvršćenim za α-podjedinične ostatke u petlji 2. Figure 4A: Binding of hCG or an hCG analog with a β-tail attached to α-subunit residues in loop 2.
Slika 4B: cAMP akumulacija hCG-a ili hCG analoga u kojih je β-rep pričvršćen na α-podjedinične ostatke u petlji 2. Figure 4B: cAMP accumulation of hCG or hCG analogs in which the β-tail is attached to α-subunit residues in loop 2.
Slika 5A: Vezanje hCG-a ili hCG analoga s β-repom pričvršćenim na α-podjedinične ostatke na karboksiterminalu. Figure 5A: Binding of hCG or an hCG analog with a β-tail attached to α-subunit residues at the carboxy terminus.
Slika 5B: cAMP akumulacija hCG-a ili hCG analoga u kojih je β-rep pričvršćen na α-podjedinične ostatke na karboksiterminalu. Figure 5B: cAMP accumulation of hCG or hCG analogs in which the β-tail is attached to α-subunit residues at the carboxy terminus.
Slika 6: Vezanje i signalne transdukcijske aktivnosti analoga u kojih je BLA pričvršćen uz α-podjedinični ostatak. Figure 6: Binding and signal transduction activities of analogs in which BLA is attached to the α-subunit residue.
Slika 7: Sekvence aminokiselina α-podjedinice i mutanata što posjeduju supstituirani cistein. (Obratiti pažnju, mutacije su označene velikim slovima i posebno su istaknute. One su pripremljene standardnom kazetnom mutagenezom i PCR mutagenezom, metodama koje standardne u struci.) (SEQ ID NO:1 - SEQ ID NO:35) Figure 7: Amino acid sequences of the α-subunit and mutants possessing a substituted cysteine. (Note that mutations are marked with capital letters and are highlighted. They were prepared by standard cassette mutagenesis and PCR mutagenesis, methods standard in the art.) (SEQ ID NO:1 - SEQ ID NO:35)
Slika 8: Sekvence aminokiselina β-podjediničnih analoga. (Obratiti pažnju, supstituirani cistein je označen velikim slovima i posebno istaknut.) (SEQ ID NO:36 – SEQ ID NO:42) Figure 8: Amino acid sequences of β-subunit analogues. (Note that the substituted cysteine is in capital letters and highlighted.) (SEQ ID NO:36 - SEQ ID NO:42)
Slika 9A: Proteinski Čvor s čvorom pričvršćenim na karboksiterminalnom kraju proteina. Figure 9A: Protein Knot with the knot attached to the carboxy-terminal end of the protein.
Slika 9B: Proteinski Čvor s čvorom pričvršćenim na aminoterminalnom kraju proteina. Figure 9B: Protein Knot with the knot attached to the amino-terminal end of the protein.
Slika 10A: Proteinski Čvor gdje je čvor naznačen cisteinskim ostatkom. Figure 10A: Protein Knot where the knot is indicated by a cysteine residue.
Slika 10B: Proteinski Čvor gdje je čvor naznačen aminokiselinskom sekvence uključujući cisteinski ostatak fuziran za protein. Figure 10B: Protein Knot where the knot is indicated by the amino acid sequence including the cysteine residue fused to the protein.
Slika 10C: Proteinski Čvor gdje je cisteinski ostatak čvora lociran na površini proteina koji jest čvor. Figure 10C: Protein Knot where the cysteine residue of the knot is located on the surface of the knot protein.
Slika 11: Učinak Čvora na aktivnost FSH. Figure 11: Effect of Node on FSH activity.
Slika 12: Sažetak aktivnosti ukriženo vezanih analoga himera u LHR i FSHR analizama koje se odnose na CF101-109, bifunkcionalne himere bez β-podjediničnog karboksiterminala hCG-a. Figure 12: Summary of activity of cross-linked chimera analogs in LHR and FSHR assays relative to CF101-109, a bifunctional chimera lacking the β-subunit carboxy-terminus of hCG.
Slika 13: Učinak Čvora na FSH receptorsko signaliziranje. Figure 13: Effect of Node on FSH receptor signaling.
Slika 14A: Učinak Čvora na LH receptorsko signaliziranje. Figure 14A: Effect of Node on LH receptor signaling.
Slika 14B: Učinak Čvora na LH receptorsko signaliziranje. Figure 14B: Effect of Node on LH receptor signaling.
Slika 15: Aminokiselinske sekvence drugih analoga (SEQ ID NO:43- SEQ ID NO:53) Figure 15: Amino acid sequences of other analogs (SEQ ID NO:43- SEQ ID NO:53)
Slika 16: Signalna transdukcija i aktivnosti vezanja LONG i SHORT heterodimera. Figure 16: Signal transduction and binding activities of LONG and SHORT heterodimers.
Slika 17: Lutropinska aktivnost hCG analoga što posjeduju čvorove β-laktamaze. Figure 17: Lutropin activity of hCG analogs possessing β-lactamase knots.
Slika 18: cAMP akumulacija u hCG-a ili analoga kojima manjka αAsn52 oligosaharid. Figure 18: cAMP accumulation in hCG or analogs lacking the αAsn52 oligosaccharide.
Slika 19: Vezanje αK44A+hCGβ za LHR. Figure 19: Binding of αK44A+hCGβ to LHR.
Slika 20A: Vezanje hCG-a i hCG analoga αK44E,K45Q+hCGβ za LHR. Figure 20A: Binding of hCG and hCG analog αK44E,K45Q+hCGβ to LHR.
Slika 20B: LHR cAMP odgovor prema hCG-u i αK44E,K45Q+hCGβ. Figure 20B: LHR cAMP response to hCG and αK44E,K45Q+hCGβ.
Slika 21A: Vezanje hCG-a i hCG analoga αK91E+hCGβ za LHR. Figure 21A: Binding of hCG and hCG analog αK91E+hCGβ to LHR.
Slika 21B: Relativne aktivnosti hCG-a i αK91E+hCGβ u LHR cAMP akumulacijskim analizama. Figure 21B: Relative activities of hCG and αK91E+hCGβ in LHR cAMP accumulation assays.
Slika 22A: LHR cAMP odgovor prema hCG-u i αK91M+hCGβ. Figure 22A: LHR cAMP response to hCG and αK91M+hCGβ.
Slika 22B: LHR vezanje hCG-a i αK91M+hCGβ. Figure 22B: LHR binding of hCG and αK91M+hCGβ.
Slika 23: LHR vezanje hCG-a i analoga što sadrže skraćene spone. Figure 23: LHR binding of hCG and analogues containing truncated linkages.
Slika 24: LHR vezanje hCG-a i analoga što sadrži skraćenu sponu. Figure 24: LHR binding of hCG and analogues containing a truncated linker.
Slika 25: Stimulacija LHR cAMP-a s hCG-om i αN52C+hCGβ,S138C. Figure 25: Stimulation of LHR cAMP with hCG and αN52C+hCGβ,S138C.
Slika 26: LHR vezanje analoga u kojih je okrnjeni karboksiterminalni rep β-podjedinice hCG-a upotrijebljen na karboksiterminalnom kraju α-podjedinice za dodavanje čvora ostacima 96, 97 ili 98 β-podjedinice hCG-a. Figure 26: LHR binding of analogs in which the truncated carboxy-terminal tail of the β-subunit of hCG is used at the carboxy-terminal end of the α-subunit to add a knot to residues 96, 97, or 98 of the β-subunit of hCG.
Slika 27: LHR signaliziranje analoga u kojih je okrnjeni karboksiterminalni rep β-podjedinice hCG-a upotrijebljen na karboksiterminalnom kraju α-podjedinice za dodavanje čvora ostacima 98 ili 99 β-podjedinice hCG-a. Figure 27: LHR signaling analogs in which the truncated carboxy-terminal tail of the β-subunit of hCG is used at the carboxy-terminal end of the α-subunit to add a knot to residues 98 or 99 of the β-subunit of hCG.
Slika 28: LHR vezanje analoga u kojih je okrnjeni karboksiterminalni rep β-podjedinice hCG-a upotrijebljen za dodavanje čvora ostacima 95 ili 96 β-podjedinice. Figure 28: LHR binding of analogs in which the truncated carboxy-terminal tail of the β-subunit of hCG was used to add a knot to residues 95 or 96 of the β-subunit.
Slika 29: LHR signaliziranje analoga u kojih je okrnjeni karboksiterminalni rep β-podjedinice hCG-a upotrijebljen za dodavanje čvora ostacima 95 ili 96 β-podjedinice. Figure 29: LHR signaling analogs in which the truncated carboxy-terminal tail of the β-subunit of hCG was used to add a knot to residues 95 or 96 of the β-subunit.
Slika 30: LHR signaliziranje analoga u kojih je GGC rep upotrijebljen za dodavanje čvora ostatku 96 β-podjedinice u usporedbi s onim što mu manjka rep i onim u kojeg je okrnjeni karboksiterminalni rep β-podjedinice hCG-a upotrijebljen za dodavanje čvora ostatku 96 β-podjedinice hCG/hFSH himera. Figure 30: LHR signaling of analogs in which the GGC tail was used to add a knot to residue 96 of the β-subunit compared to one lacking the tail and one in which the carboxy-terminal tail of the hCG β-subunit was used to add a knot to residue 96 of the β-subunit. subunits of the hCG/hFSH chimera.
Slika 31: LHR signaliziranje analoga u kojih je okrnjeni karboksiterminalni rep β-podjedinice hCG-a upotrijebljen za dodavanje čvora ostacima 98 ili 99 β-podjedinice. Figure 31: LHR signaling analogs in which the truncated carboxy-terminal tail of the β-subunit of hCG was used to add a knot to residues 98 or 99 of the β-subunit.
Slika 32: LHR signaliziranje analoga što pokazuje utjecaj repa na dodavanje čvora ostatku 95 β-podjedinice hCG-a i na analog kojem nedostaje sposobnost glikoziliranja ostatka 52 α-podjedinice. Figure 32: LHR signaling analogs showing the effect of tailing on knot addition to residue 95 of the β-subunit of hCG and to an analog lacking the ability to glycosylate residue 52 of the α-subunit.
Slika 33: cAMP odgovor na ukriženo vezane analoge. Figure 33: cAMP response to cross-linked analogs.
Detaljni opis izuma Detailed description of the invention
Trenutačno, većina označenih proteina je pripravljena kao fuzijski proteini sa oznakom dodanom na aminoterminalni ili karboksiterminalni kraj proteina ili sa nekoliko ostataka umetnutih u proteinsku petlju. Dok terminalne oznake mogu biti bilo koje veličine, oznake umetnute u proteinsku petlju su obično limitirane na relativno malo aminokiselinskih ostataka osim ako su umetnute između proteinskih domena. Proteini isto tako mogu biti označeni na različitim mjestima kroz uvođenje cisteina na mjesto što se označava i onda reagiranjem sulfhidrilne skupine cisteina sa sulfhidril-specifičnim reagensom. Međutim,ovo može biti teško ostvarivo kada protein sadrži druge cisteine ili kad je pripravljen pod uvjetima koji uzrokuju «blokadu» cisteina, kao što je često slučaj kad su proteini eksprimirani u eukariotskim stanicama. Currently, most tagged proteins are prepared as fusion proteins with a tag added to the amino-terminal or carboxy-terminal end of the protein or with a few residues inserted into the protein loop. While terminal tags can be of any size, tags inserted into a protein loop are usually limited to relatively few amino acid residues unless inserted between protein domains. Proteins can also be labeled at different sites by introducing cysteine into the site to be labeled and then reacting the sulfhydryl group of the cysteine with a sulfhydryl-specific reagent. However, this can be difficult to achieve when the protein contains other cysteines or when it is prepared under conditions that cause cysteine "blockage", as is often the case when proteins are expressed in eukaryotic cells.
Predloženi izum osigurava poboljšane metode za etiketiranje ili označavanje proteina. Metode predloženog izuma izbjegavaju komplikacije povezane sa etiketiranjem ili označavanjem pomoću uvođenja cisteina, kao što je gore opisano, i dozvoljavaju testerima ili čvorovima različitih veličina da budu pričvršćeni na površinu proteina i drugdje osim na njegovim terminalnim krajevima. Predloženi izum osigurava metode što dodaju «čvorove» raznolikih veličina na definirane lokacije na površini hCG-a. Čvorovi mogu biti mali poput jednog cisteinskog ostatka. Čvorovi mogu biti kratki peptidi, poput ostataka što okružuju ostatak 138 β-podjedinice hCG-a. Čvorovi isto tako mogu biti veliki kao cijeli protein. Na primjer, β-laktamaza može biti upotrijebljena kao čvor. Čvorovi se dodaju za vrijeme sinteze proteina čineći nepotrebnim uklanjanje bilo kakvih blokirajućih ostataka ili pričvršćivanje kakvih protektivnih skupina, što su zadaci koji mogu uzrokovati proteinsku denaturaciju. The present invention provides improved methods for labeling or labeling proteins. The methods of the present invention avoid the complications associated with labeling or tagging by introducing cysteines, as described above, and allow probes or knots of different sizes to be attached to the surface of the protein elsewhere than at its terminal ends. The proposed invention provides methods that add "nodes" of various sizes to defined locations on the surface of hCG. Knots can be as small as a single cysteine residue. The knots can be short peptides, such as the residues surrounding residue 138 of the β-subunit of hCG. Nodules can also be as large as the entire protein. For example, β-lactamase can be used as a node. The knots are added during protein synthesis, making it unnecessary to remove any blocking residues or attach any protective groups, tasks that can cause protein denaturation.
Predloženi izum primjenjuje novu strategiju za pričvršćivanje širokog raspona testera ili oznaka za specifična mjesta na proteinskoj površini bazirajući se na otkriću da je fleksibilni β-podjedinični rep sposoban da se ukriženo veže za jedan od nekoliko α-podjediničnih ostataka što su zamijenjeni cisteinom. Čvorovi mogu biti fluorescentni protein, kao što je zeleni fluorescentni protein ili srodna molekula. Mogu imati sposobnost da vežu druge molekule, što su osobine nađene u liganda ili receptora. Oni mogu biti proteaze, toksini, antitijela ili fragmenti antitijela, sekvence kao što su nađene u TAT proteina humanog virusa imunodeficijencije koje omogućuju proteinima da budu translatirani preko membrana, nukleinske kiseline, ili oligosaharidi. The proposed invention applies a novel strategy for attaching a wide range of probes or labels to specific sites on the protein surface based on the discovery that a flexible β-subunit tail is capable of cross-linking to one of several α-subunit residues that are replaced by cysteine. The nodes can be a fluorescent protein, such as green fluorescent protein or a related molecule. They may have the ability to bind other molecules, which are properties found in ligands or receptors. They may be proteases, toxins, antibodies or antibody fragments, sequences such as those found in the TAT protein of the human immunodeficiency virus that enable proteins to be translated across membranes, nucleic acids, or oligosaccharides.
U jednom ostvarenju, kompozicija koja se sastoji od proteinske porcije, gdje proteinska porcija sadrži supstituirani cisteinski ostatak na željenoj lokaciji za označavanje, porcije repa na terminalnom kraju proteinske porcije; i čvora, gdje je čvor lociran na slobodnom terminalnom kraju porcije repa i sadrži cisteinski ostatak, i gdje cisteinski ostatak čvora formira disulfid sa supstituiranim cisteinom u proteinskoj porciji. Termin «proteinska porcija» se odnosi na bilo koji protein ili polipeptid. Termin «porcija repa» se odnosi na niz aminokiselina dostatne dužine da dozvoli cisteinu u čvoru da formira disulfid sa supstituiranim cisteinom u proteinskoj porciji. Porcija repa može se sastojati od nativne polipeptidne porcije proteina proteinske porcije, kao što karboksiterminal β-podjedinice to čini u hCG-u (vidi Sliku 1), ili se može sastojati od ne-nativnog polipeptida dodanog na terminalni kraj proteinske porcije. Porciji repa bi također trebali nedostajati ostaci koji bi spriječili čvor od pričvršćivanja za supstituirani cistein, kao što su transmembranske domene ili ostaci koji će stvoriti mjesto za vezanje ostalih proteina. Termin «čvor» se odnosi na cistein i bilo koje ostatke na obje strane cisteina koji su locirani u blizini slobodnog terminalnog kraja repa. Čvor može uključivati samostalan cisteinski ostatak, linearan niz aminokiselina koje sadrže cistein, linearan niz aminokiselina fuziranih za protein, gdje je cistein lociran u nizu aminokiselina, ili protein koji sadrži cisteinski ostatak na svojoj površini. Čvor može biti iskonstruiran za posebnu svrhu ili upotrebu rezultirajući kustomiziranom oznakom ili testerom. Na primjer, čvor može biti oznaka epitopa, signalna sekvenca, sekvenca sa visokom specifičnošću za kuglice na purifikacijskoj kolumni, enzim, ili ciljani protein. In one embodiment, a composition comprising a protein portion, wherein the protein portion contains a substituted cysteine residue at a desired location for labeling, a tail portion at the terminal end of the protein portion; and a knot, wherein the knot is located at the free terminal end of the tail portion and contains a cysteine residue, and wherein the cysteine residue of the knot forms a disulfide with a substituted cysteine in the protein portion. The term "protein portion" refers to any protein or polypeptide. The term "tail portion" refers to a sequence of amino acids of sufficient length to allow the cysteine in the knot to form a disulfide with a substituted cysteine in the protein portion. The tail portion may consist of the native polypeptide portion of the protein portion of the protein portion, as the carboxyterminal β-subunit does in hCG (see Figure 1), or it may consist of a non-native polypeptide added to the terminal end of the protein portion. The tail portion should also lack residues that would prevent the knot from attaching to the substituted cysteine, such as transmembrane domains or residues that would create a binding site for other proteins. The term "knot" refers to the cysteine and any residues on either side of the cysteine that are located near the free terminal end of the tail. The knot may include a stand-alone cysteine residue, a linear sequence of amino acids containing cysteine, a linear sequence of amino acids fused to a protein where the cysteine is located in the sequence of amino acids, or a protein containing a cysteine residue on its surface. A node can be engineered for a specific purpose or use resulting in a customized tag or tester. For example, a node can be an epitope tag, a signal sequence, a sequence with high specificity for beads on a purification column, an enzyme, or a target protein.
U slijedećem ostvarenju izuma, metode za označavanje proteina na specifičnom mjestu su obznanjene. Metode uključuju selekcioniranje željenog proteina, lociranje mjesta na željenom proteinu koje će biti označeno, te selekcioniranje željenog čvora. Željeni čvor mora sadržavati cisteinski ostatak. Metode nadalje uključuju pripremanje konstrukcije što enkodira željeni protein, porciju repa i željeni čvor. Željeni protein što je enkodiran s konstrukcijom uključuje cisteinski ostatak supstituiran na mjestu koje će biti označeno. Konstrukcija je tada umetnuta u stanicu za ekspresiju označenog proteina, gdje cistein u čvoru i supstituirani cistein u željenom proteinu formiraju disulfidnu vezu. In another embodiment of the invention, methods for labeling proteins at a specific site are disclosed. The methods include selecting a desired protein, locating a site on the desired protein to be labeled, and selecting a desired node. The desired knot must contain a cysteine residue. The methods further include preparing a construct that encodes the desired protein, the tail portion, and the desired node. The desired protein encoded by the construct includes a cysteine residue substituted at the site to be labeled. The construct is then inserted into a cell to express the tagged protein, where the cysteine in the junction and the substituted cysteine in the desired protein form a disulfide bond.
Termin «konstrukcija» se odnosi na vektor nukleinske kiseline koji se sastoji od promotera koji je povezan sa ekspresijskom kazetom konstruiranom da enkodira poseban poteinski produkt. Konstrukcija nadalje uključuje sve neophodne sekvence tako da enkodirani protein može biti eksprimiran i bilo koje sekvence koje mogu biti uključene da kontroliraju ekspresiju kazete. Ove sekvence mogu uključivati, ali nisu na to ograničene, promoter ili inicijacijsku sekvencu, unaprijeđujuću sekvencu, terminirajuću sekvencu, RNA procesirajuće signale, i/ili poliadenilacijsku sekvencu. Termin «neophodne sekvence za ekspresiju ekspresijske kazete» se odnosi na sekvence potrebne da osiguraju RNA transkripciju i posljedičnu translaciju ekspresivne kazete za produkciju proteinskog produkta. Termin «promoter» se odnosi na DNA sekvencu koja je vezana RNA polimerazom i potrebna je za iniciranje RNA transkripcije gena. Postoji velik broj promotera koji su poznati u struci, uključujući one koji mogu unaprijediti ili kontrolirati ekspresiju gena ili ekspresijsku kazetu. Konstrukcije ovog izuma mogu biti modificirane PCR-om i kazetnom mutagenezom za stvaranje konstrukcije koja enkodira željeni proteinski čvor. The term "construct" refers to a nucleic acid vector consisting of a promoter linked to an expression cassette engineered to encode a specific protein product. The construct further includes all necessary sequences so that the encoded protein can be expressed and any sequences that may be included to control expression of the cassette. These sequences may include, but are not limited to, a promoter or initiation sequence, an enhancer sequence, a termination sequence, RNA processing signals, and/or a polyadenylation sequence. The term "necessary sequences for the expression of an expression cassette" refers to sequences necessary to ensure RNA transcription and subsequent translation of the expression cassette for the production of a protein product. The term "promoter" refers to a DNA sequence that is bound by RNA polymerase and is required to initiate RNA transcription of a gene. There are a large number of promoters known in the art, including those that can promote or control the expression of a gene or an expression cassette. The constructs of the present invention can be modified by PCR and cassette mutagenesis to create a construct that encodes the desired protein node.
Čvorovi na specifičnom mjestu na proteinu mogu se koristiti da testiraju distancu ispitivanih ostataka do interface-a receptora ili drugog proteina. Na primjer, čvorovi mogu biti korisno upotrijebljeni da determiniraju blizinu α-podjediničnih ostataka do mjesta vezanja receptora u hCG-u. Pričvršćivanje testera za ostatke koji su locirani na vežućem džepu bi poništilo vežuću akivnost upućujući na to da je oznaka locirana na vežućem džepu. Dodatna prednost upotrebe čvorova na specifičnom mjestu jest ta da veći testeri mogu biti iskorišteni za identifikaciju ostataka koji su blizu protein-receptorskog interface-a. Ova testirajuća strategija također može biti iskorištena za dodavanje epitopnih oznaka ili signalnih sekvenca bilo kojem željenom mjestu na površini proteina. Dodatno, proteinski čvorovi također mogu biti u velikoj mjeri upotrijebljeni za proteinsku imobilizaciju ili proteinsko ciljanje. Ukoliko je mjesto prepoznavanja proteaze iskonstruirano unutar fleksibilnog repa, tada rep može biti otcijepljen nakon ostvarenja ukrižene veze, ostavljajući tester sapet disulfidom za površinu proteina, ali ne i sapet repom. Nodes at a specific location on a protein can be used to test the distance of the investigated residues to the interface of a receptor or another protein. For example, knots can be usefully used to determine the proximity of α-subunit residues to the receptor binding site in hCG. Attachment of the probe to residues located on the binding pocket would nullify the binding activity indicating that the label is located on the binding pocket. An additional advantage of using site-specific nodes is that larger probes can be used to identify residues close to the protein-receptor interface. This assay strategy can also be used to add epitope tags or signal sequences to any desired location on the protein surface. Additionally, protein assemblies can also be extensively used for protein immobilization or protein targeting. If the protease recognition site is constructed within a flexible tail, then the tail can be cleaved after cross-linking, leaving the probe disulfide-tethered to the protein surface, but not tail-tethered.
Upotrebe proteinskih čvorova producirale su upotrebu metoda izuma, a nisu ograničene samo na zaključivanje o distanci između mjesta na proteinima. Ako je željena ciljana proteaza, mnoge različite proteaze bi mogle biti pričvršćene ili na aminoterminal ili karboksiterminal proteina jednostavno fuzirajući kodirajuću sekvencu proteaze za 5' ili 3' kraj kodirajuće sekvence proteina za modificiranje kao što je već bilo opisano za pripremanje fuzijskih proteina (U.S. Patent 6,300,099, Sledziewski et al.). Na nesreću, blizina proteaze proteinu može rezultirati destrukcijom proteina. The uses of protein knots have produced the use of the methods of the invention, and are not limited to the inference of distances between sites on proteins. If a targeted protease is desired, many different proteases could be attached to either the amino- or carboxy-terminal of the protein simply by fusing the protease coding sequence to the 5' or 3' end of the protein coding sequence to be modified as previously described for the preparation of fusion proteins (U.S. Patent 6,300,099 , Sledziewski et al.). Unfortunately, the proximity of the protease to the protein can result in protein destruction.
Upotreba proteinskih čvorova rješava ovaj problem zato što upotrebljavajući strategiju za pričvršćivanje čvora proteinu kao što je ovdje opisano, proteaza može biti zadržana u poziciji koja ju onemogućava da napadne molekulu za koju je pričvršćena. Nadalje, orijentacija proteaze može joj olakšati kataliziranje cijepanja željenog supstrata, kao što je receptor. Proteaza može biti iskonstruirana na hCG-u koristeći metode ovdje opisane tako da bi proteaza cijepala LH receptor preferencijalno bilo kojem drugom proteinu, rezultirajući gubitkom lutropinske aktivnosti. Ova protein čvor proteaza može biti korištena u terapijski željene svrhe za tretiranje sindroma policističnih jajnika, uzroka skoro 1⁄3 cjelokupnog humanog infertiliteta. The use of protein knots solves this problem because by using a strategy for attaching a knot to a protein as described herein, the protease can be held in a position that prevents it from attacking the molecule to which it is attached. Furthermore, the orientation of the protease may make it easier for it to catalyze the cleavage of a desired substrate, such as a receptor. A protease can be engineered on hCG using the methods described herein such that the protease cleaves the LH receptor preferentially over any other protein, resulting in loss of lutropin activity. This protein knot protease can be used for therapeutically desired purposes for the treatment of polycystic ovary syndrome, the cause of almost 1⁄3 of all human infertility.
U slijedećem ostvarenju izuma, kompozicije i metode ovog izuma mogu biti iskorištene u promicanju stabilne asocijacije dvaju proteina. Ovdje pribavljeni podaci pokazuju da su ukriženo vezani proteinski analozi hCG-a mnogo stabilniji nego nativni hCG pri niskim vrijednostima pH. Uvođenje interpodjediničnih disulfida u hCG povećava stabilnost heterodimera (Matzuk, M. M. & Boime, I. (1988) J.Cell.Biol. 106, 1049-1059; Heikoop, J. C.; van den Boogaart; Mulders, J. W.; Grootenhuis, P. D., (1997), Structure-based design and protein engineering of intersubunit disulfide bonds in gonadotropins, Nature Biotechnology 15: 658-662). Prethodno, interpodjedinične disulfidne veze su bile uvedene u protein bazirajući se na njegovoj kristalnoj strukturi. Predloženi izum obznanjuje metode za uvođenje interpodjediničnih disulfida u dva proteina tijekom njihove sinteze kad struktura visoke rezolucije nije raspoloživa. In another embodiment of the invention, the compositions and methods of this invention can be used to promote a stable association of two proteins. The data obtained here show that cross-linked protein analogues of hCG are much more stable than native hCG at low pH values. Introduction of intersubunit disulfides into hCG increases heterodimer stability (Matzuk, M. M. & Boime, I. (1988) J.Cell.Biol. 106, 1049-1059; Heikoop, J. C.; van den Boogaart; Mulders, J. W.; Grootenhuis, P. D., (1997) ), Structure-based design and protein engineering of intersubunit disulfide bonds in gonadotropins, Nature Biotechnology 15: 658-662). Previously, intersubunit disulfide bonds were introduced into a protein based on its crystal structure. The proposed invention discloses methods for introducing intersubunit disulfides into two proteins during their synthesis when a high-resolution structure is not available.
U slijedećem ostvarenju izuma, kompozicije i metode mogu biti iskorištene u promicanju stabilne asocijacije DNA polimeraze za DNA. Očekiva se da će uvođenje spone koja se omata oko DNA i stabilizirana je za polimerazu disulfidnom vezom stabilizirati polimerazu za DNA, tako povećavajući duljinu rezultirajuće transkripcije. In another embodiment of the invention, the compositions and methods may be used to promote stable association of DNA polymerase to DNA. The introduction of a linker that wraps around the DNA and is stabilized for the polymerase by a disulfide bond is expected to stabilize the polymerase for the DNA, thus increasing the length of the resulting transcription.
U dodatnom ostvarenju izuma, kompozicije i metode izuma mogu biti iskorištene za produkciju proteinskih heterodimera glikoproteinskih hormona kojima nedostaje jedan ili više njihovih oligosaharida. Eliminacija glikozilacijskog signala na petlji 2 α-podjedinice hCG-a reducira sposobnost stanica sisavaca da secerniraju heterodimere i izazovu biološki odgovor (Einstein, M., Lin, W., Macdonald, G. J. & Moyle, W. R. (2001) Exp. Biol. Med. 226, 581-590; Slaughter, S., Wang, Y. H., Myers, R. V. & Moyle, W. R. (1995) Moll. Cell. Endocrinol. 112, 21-25; Yen, S. S. C., Llierena, O., Little, B. & Parsons, O. H. (1968) J. Clin. Endocrinol. Metab. 28, 1763-1767, Matzuk, M. M.; Boime, I., (1989), Mutagenesis and gene transfer define site-specific roles of gonadotropin oligosaccharides. Biol. Reprod. 40:48-53). Kao što je ovdje prikazano, ko-ekspresija hCGβS138C i αN52C, analoga α-podjedinice u kojeg je cistein supstituiran umjesto ostatka Asn52 α-podjedinice, omogućuje produkciju heterodimera u količinama usporedivim sa hCG-om. Ova mutacija α-podjedinice je eliminirala njezin glikozilacijski signal i dovela do hCG analoga koji je imao znatno veću efikasnost nego analog hCG-a u kojeg je oligosaharid petlje 2 α-podjedinice eliminiran glikanaznom digestijom. In an additional embodiment of the invention, the compositions and methods of the invention can be used for the production of protein heterodimers of glycoprotein hormones that lack one or more of their oligosaccharides. Elimination of the glycosylation signal on loop 2 of the hCG α-subunit reduces the ability of mammalian cells to secrete heterodimers and elicit a biological response (Einstein, M., Lin, W., Macdonald, G. J. & Moyle, W. R. (2001) Exp. Biol. Med. 226, 581-590; Slaughter, S., Wang, Y. H., Myers, R. V. & Moyle, W. R. (1995) Moll. Cell. Endocrinol. 112, 21-25; Yen, S. S. C., Llierena, O., Little, B. & Parsons, O. H. (1968) J. Clin. Endocrinol. Metab. 28, 1763-1767, Matzuk, M. M.; Boime, I., (1989), Mutagenesis and gene transfer define site-specific roles of gonadotropin oligosaccharides. Biol. Reprod. 40:48-53). As shown here, co-expression of hCGβS138C and αN52C, an analog of the α-subunit in which cysteine is substituted for the Asn52 residue of the α-subunit, allows the production of heterodimers in amounts comparable to hCG. This mutation of the α-subunit eliminated its glycosylation signal and led to an hCG analog that had significantly higher efficiency than the hCG analog in which the oligosaccharide of loop 2 of the α-subunit was eliminated by glycanase digestion.
U dodatnom ostvarenju, metode za upotrebu kompozicija i metoda ovog izuma za promicanje formacije proteinskih multimera u kojima podjedinice imaju malo ili nemaju afiniteta jedna prema drugoj su obznanjene. Na primjer, pribavljeni podatak na Slici 6 ilustrira da je moguće pričvrstiti enzim β-laktamazu za hCG na nekoliko različitih mjesta. Nije poznato da β-laktamaza stupa u asocijaciju sa hCG-om. Uvođenjem mjesta za cijepanje proteazom, između β-podjedinice hCG-a i cisteina na sigurnosnom pojasu, što cijepa karboksiterminal β-podjedinice, bilo bi moguće pripremiti hetrotrimere u kojima su β-laktamaza ili ostali proteini čvrsto pričvršćeni za gotovo svako mjesto na hCG-u. In an additional embodiment, methods for using the compositions and methods of the present invention to promote the formation of protein multimers in which the subunits have little or no affinity for each other are disclosed. For example, the data obtained in Figure 6 illustrates that it is possible to attach the β-lactamase enzyme to hCG at several different sites. β-lactamase is not known to associate with hCG. By introducing a protease cleavage site, between the β-subunit of hCG and the seat belt cysteine, which cleaves the carboxy-terminal β-subunit, it would be possible to prepare heterotrimers in which β-lactamase or other proteins are firmly attached to almost any site on hCG .
U drugom ostvarenju, cistein na čvoru može biti pomaknut na mjesto na površini porcije čvora. Ovo bi dozvolilo direktno pričvršćivanje čvora na protein u željenoj orijentaciji i željenom mjestu na proteinu. In another embodiment, the cysteine on the knot may be displaced to a site on the surface of the knot portion. This would allow direct attachment of the knot to the protein in the desired orientation and location on the protein.
U drugom ostvarenju, metode ovog izuma mogu biti iskorištene za označavanje proteina s epitopnim oznakama. Epitopne oznake su često pričvršćene za proteine da olakšaju detekciju proteinsko-proteinskih ili proteinsko-makromolekularnih interakcija. U prošlosti, epitopne oznake su bile pričvršćene za aminoterminalni ili karboksiterminalni kraj proteina. Ipak mnoge epitopne oznake funkcioniraju samo na jednom kraju proteina. Nadalje, korisnost epitopnih oznaka dodanih na krajeve proteina je značajno smanjena kad su krajevi proteina uključeni u proteinsku funkciju. Metode ovog izuma dozvoljavaju lokalizaciju epitopnih oznaka na drugim mjestima, a ne samo na krajevima proteina, čineći epitopne oznake puno korisnijim. Uvođenje mjesta za cijepanje u porciju repa bi oslobodilo kraj proteina bez disrupcije epitopne oznake. In another embodiment, the methods of the present invention can be used to label proteins with epitope tags. Epitope tags are often attached to proteins to facilitate the detection of protein-protein or protein-macromolecular interactions. In the past, epitope tags were attached to the amino-terminal or carboxy-terminal ends of proteins. However, many epitope tags function only at one end of the protein. Furthermore, the utility of epitope tags added to protein ends is significantly reduced when the protein ends are involved in protein function. The methods of the present invention allow epitope tags to be localized at other sites than just the ends of proteins, making epitope tags much more useful. Introducing a cleavage site into the tail portion would release the end of the protein without disrupting the epitope tag.
U slijedećem ostvarenju izuma, aldehidni ostaci mogu biti uvedeni na specifična mjesta u proteine. Aldehidi su vrlo poželjne reaktivne skupine koje se normalno ne nalaze u proteinima i mogu se koristiti za pričvršćivanje nekoliko različitih reagensa kao što su fluorofore na površinu proteina. Ova procedura izvlači korist od dobro poznate reaktivnosti aminoterminalnih ostataka serina ili treonina u blagoj perodatum oksidaciji (Yoo, J., Ji., I. & Ji., T. H. (1991) J. Biol. Chem. 266, 17741-17743; Geoghegan, K. F.; Stroh, J.G., (1992) Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alchol. Application to modification at N-terminal serine. Bioconjug. Chem. 3:138-146). Odatle, serinski ostatak može biti uveden odmah nakon enzimskog cijepanja mjesta na proteinu. Na primjer, mjesto prepoznato od enterokinaze može biti odmah uvedeno aminoterminalno od serina, što bi bilo aminoterminal cisteina za upotrebu ukriženog vezanja čvora za ciljani cistein na mjestu za označavanje. Ovo bi rezultiralo sekvencom Xl-Asp-Asp-Asp-Asp-Lys-Ser-Ym-Cys-Zn (SEQ ID NO:56) gdje se X, Y i Z odnose na bilo koje aminokiseline porcije repa i l, m, i n se odnose na duljine aminokiselina porcije repa. Produkcija proteina bi rezultirala cisteinom u porciji repa koji postaje ukriženo vezan sa željenim mjestom na proteinu, koje je promijenjeno u cistein. Cijepanje s enterokinazom bi rezultiralo stvaranjem aminoterminalnog serina koji je lako oksidiran s blagim periodatum tretmanom. Aldehid koji je proizveden može lako reagirati sa nekom od mnoštva hidriranih deriviranih mješavina uključujući različite fluorofore i biotin. Ova metoda bila bi osobito korisna u proteina koji ne sadrže nevezane cisteine. In another embodiment of the invention, aldehyde residues can be introduced at specific locations in proteins. Aldehydes are highly desirable reactive groups not normally found in proteins and can be used to attach several different reagents such as fluorophores to the protein surface. This procedure takes advantage of the well-known reactivity of amino-terminal serine or threonine residues in mild perodatum oxidation (Yoo, J., Ji., I. & Ji., T. H. (1991) J. Biol. Chem. 266, 17741-17743; Geoghegan, K. F.; Stroh, J.G., (1992) Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alchol. Application to modification at N-terminal serine. Bioconjug. Chem. 3:138-146). From there, a serine residue can be introduced immediately after enzymatic cleavage of the site on the protein. For example, the enterokinase recognition site can be introduced immediately amino-terminal to the serine, which would be the amino-terminal cysteine to use a cross-linking knot to the target cysteine at the labeling site. This would result in the sequence Xl-Asp-Asp-Asp-Asp-Lys-Ser-Ym-Cys-Zn (SEQ ID NO:56) where X, Y, and Z refer to any amino acids of the tail portion and l, m, and n are related to the amino acid lengths of the tail portion. Production of the protein would result in a cysteine in the tail portion becoming cross-linked to the desired site on the protein, which is changed to a cysteine. Cleavage with enterokinase would result in the formation of an amino-terminal serine that was readily oxidized with mild periodatum treatment. The aldehyde that is produced can readily react with any of a number of hydrogenated derivatized mixtures including various fluorophores and biotin. This method would be particularly useful in proteins that do not contain unbound cysteines.
U dodatnom ostvarenju izuma, metode za upotrebu kompozicija ovog izuma za blokiranje specifičnih mjesta na proteinima su obznanjene. Na primjer, bilo bi poželjno blokirati aktivno mjesto enzima ili toksina koji ima potencijal da ubija stanice dok aktivnost ne postane željena. Terapija raka može biti odlično područje za upotrebu kompozicija i metoda ovog izuma. Prema tome, cistein može biti uveden u enzim ili toksin na mjestu u blizini aktivnog mjesta enzima ili toksina. Dodatak čvora koji sadrži cistein na aminoterminalni ili karboksiterminalni kraj proteina koji ima sposobnost formiranja disulfida s cisteinom u blizini aktivnog mjesta bi uzrokovalo da čvor spriječi aktivno mjesto od interakcije s njegovim ciljem. Čvor se može sastojati od ciljanog proteina fuziranog za za kraj porcije repa što bi omogućilo kompleksu enzim/toksin – ciljani protein da se usidri sa specifičnim ciljem na površinu stanice. Tretiranje kompleksa proteazom koja cijepa porciju repa bi izložilo aktivno mjesto enzima ili toksina. Ova strategija bi mogla biti iskorištena da sakrije aktivnost enzima ili toksina dok ne dosegne mjesto koje sadrži enzim koji iscijepa porciju repa, tako razotkrivajući toksin. Na primjer, ovo se može događati nakon internalizacije kopleksa u stanicu. In an additional embodiment of the invention, methods for using the compositions of this invention to block specific sites on proteins are disclosed. For example, it would be desirable to block the active site of an enzyme or toxin that has the potential to kill cells until the activity becomes desired. Cancer therapy may be an excellent area for the use of the compositions and methods of this invention. Therefore, cysteine can be introduced into an enzyme or toxin at a site near the active site of the enzyme or toxin. Addition of a cysteine-containing knot to the amino-terminal or carboxy-terminal end of a protein that has the ability to form a disulfide with a cysteine near the active site would cause the knot to prevent the active site from interacting with its target. The knot may consist of a target protein fused to the end of a tail portion that would allow the enzyme/toxin-target protein complex to anchor with a specific target to the cell surface. Treatment of the complex with a protease that cleaves the tail portion would expose the active site of the enzyme or toxin. This strategy could be used to hide the activity of an enzyme or toxin until it reaches a site containing an enzyme that cleaves a portion of the tail, thus exposing the toxin. For example, this may occur after internalization of the complex into the cell.
Dodatna upotreba čvora bila bi ta da spriječi nepoželjne asocijacije između proteina koji normalno stvaraju kompleks jedan s drugim. Prema tome, disulfid bi bio konstruiran da drži čvor u poziciji na interface-u između dva proteina. An additional use of the knot would be to prevent unwanted associations between proteins that normally form a complex with each other. Therefore, the disulfide would be designed to hold the knot in position at the interface between the two proteins.
U dodatnom ostvarenju, predloženi izum osigurava metodu za preciznu separaciju ciljanog proteina u samo nekoliko koraka. Upotrebljavajući ovdje opisane metode, stvorena je ekspresijska konstrukcija što enkodira protein od interesa, porciju repa i čvor. Lokacija ciljanog cisteinskog ostatka na proteinu od interesa može biti na karboksiterminalnom kraju, aminoterminalnom kraju, na bilo kojoj željenoj lokaciji na površini proteina od interesa. Jednom kad se sparivajući cistein na čvoru veže za cistein na željenom proteinu nakon konstrukcijske ekspresije, rezultirajući kompleks protein-čvor je brzo pregledan u kolumni. Uz dobar izbor čvrsto-vežućeg čvora, kompleks protein-čvor veže se za privlačivu smolu i nevezani proteini i celularne komponenete su isprani. Zatim, kompleks je eluiran, čvor je otcijepljen, i samo purificirani protein ostaje. Na primjer (Strategeneov afinitet™) pCAL vektor može biti iskorišten i kalmodulin-vežući peptid (KVP) može biti izabran kao čvor. KVP-čvor se veže za kalmodulinsku smolu i može biti eluiran sa 2mM EDTA pri neutralnom pH, tako izbjegavajući stroge eluacijske uvjete što mogu denaturirati proteine. Ipak, postoje mnoge moguće kombinacije vektora i čvora koje mogu biti korisno upotrijebljene predloženim izumom. In an additional embodiment, the present invention provides a method for the precise separation of a target protein in just a few steps. Using the methods described herein, an expression construct was created that encodes the protein of interest, the tail portion, and the node. The location of the target cysteine residue on the protein of interest can be at the carboxy-terminal end, the amino-terminal end, any desired location on the surface of the protein of interest. Once the pairing cysteine on the knot binds to a cysteine on the desired protein after construct expression, the resulting protein-knot complex is rapidly screened on the column. With a good choice of tight-binding knot, the protein-knot complex binds to the attractant resin and unbound proteins and cellular components are washed away. Then, the complex is eluted, the knot is cleaved, and only the purified protein remains. For example (Strategene's Affinity™) pCAL vector can be used and calmodulin-binding peptide (KVP) can be selected as a node. KVP-knot binds to calmodulin resin and can be eluted with 2mM EDTA at neutral pH, thus avoiding harsh elution conditions that can denature proteins. However, there are many possible combinations of vectors and nodes that may be usefully employed by the proposed invention.
U daljnjem aspektu izuma koji se odnosi na proteinsku purifikaciju, protein od interesa može biti konstruiran tako da proteinski čvor ima kratki rep sa mjestom cijepanja. Rep čvora mora biti dovoljno kratak tako da čvor ne može stvoriti disulfidni spoj sa samim proteinom. Umjesto toga, struktura proteina i repa su takve da čvor pridonosi stvaranju disulfidne veze s drugim proteinom. U soluciji, proteini s kratkim repovima bi se poredali kao niz kuglica, povezani disulfidnom vezom između cisteina na čvoru prvog proteina i cisteina na proteinskoj porciji drugog proteina. Čvor na drugom proteinu bi tada formirao disulfidnu vezu sa cisteinom na proteinskoj porciji trećeg proteina i tako dalje. Rezultirajući lanac proteina može putovati sukroznim gradijentom i biti centrifugiran, lanac bi pao na dno gradijenta zahvaljujući svojoj težini. Napokon, za separaciju proteinskih lanaca od ostalog teškog materijala kao što su membrane liziranih stanica, najniži sloj gdje se lanci nalaze bio bi tretiran s enzimom specifičnim za mjesto cijepanja na kratkom repu, rezultirajući sa individualnim proteinima. Mješavina bi tad bila recentrifugirana. Individualni proteini bi ostali u blizini vrha gradijenta, dozvoljavajući lako uklanjanje i purifikaciju. Ovaj lanac proteina bi isto tako mogao biti purificiran upotrebom drugih metoda koje su gore raspravljene ili drugim metodama za proteinsku purifikaciju znanim u struci. In a further aspect of the invention relating to protein purification, the protein of interest can be engineered such that the protein knot has a short tail with a cleavage site. The tail of the knot must be short enough so that the knot cannot form a disulfide bond with the protein itself. Instead, the structure of the protein and the tail are such that the knot contributes to the formation of a disulfide bond with another protein. In solution, the short-tailed proteins would line up as a series of beads, linked by a disulfide bond between a cysteine on the node of the first protein and a cysteine on the protein portion of the second protein. The knot on the second protein would then form a disulfide bond with the cysteine on the protein portion of the third protein, and so on. The resulting protein chain can travel down the sucrose gradient and be centrifuged, the chain would fall to the bottom of the gradient due to its weight. Finally, to separate protein chains from other heavy material such as lysed cell membranes, the lowest layer where the chains reside would be treated with an enzyme specific for the short tail cleavage site, resulting in individual proteins. The mixture would then be recentrifuged. Individual proteins would remain near the top of the gradient, allowing easy removal and purification. This protein chain could also be purified using the other methods discussed above or other protein purification methods known in the art.
U drugom aspektu izuma, protein od interesa može biti konstruiran tako da svaki proteinski čvor uzajamno djeluje s drugim proteinskim čvorom formirajući rešetki sličnu strukturu. Repovi bi trebali biti konstruirani tako da je čvor onemogućen da reagira sa cisteinom na proteinskoj porciji, slično repovima u metodi bisernog niza. Proteini se isto tako mogu sastojati od repova i čvorova na oba terminalna kraja proteina. Proteinska porcija bi trebala sadržavati multiple supstitucijske cisteinske ostatke tako da čvorovi iz više od jednog separiranog proteina mogu formirati disulfidne veze sa proteinom. Broj i lokacija supstituiranih cisteina na proteinskim čvorovima bi trebale biti determinirane prije kostrukcije koristeći kompjuterski modelirajući program ili neku drugu metodu. U soluciji, proteini bi formirali rešetki sličnu strukturu kao što samostalan protein može formirati dislfidne veze s više nego jednim proteinom. Ovaj matriks proteinskih čvorova bi tada mogao biti purificiran jednom od gore navedenih metoda, imajući na umu da visoka molekularna težina strukture čini ju dobrim kandidatom za centrifugacijske tehnike. In another aspect of the invention, the protein of interest can be engineered such that each protein node interacts with another protein node to form a lattice-like structure. The tails should be constructed so that the knot is prevented from reacting with the cysteine on the protein portion, similar to the tails in the pearl string method. Proteins can also consist of tails and knots at both terminal ends of the protein. The protein portion should contain multiple substitutional cysteine residues so that knots from more than one separated protein can form disulfide bonds with the protein. The number and location of substituted cysteines on protein nodes should be determined before construction using a computer modeling program or some other method. In solution, proteins would form a lattice-like structure just as an individual protein can form disulfide bonds with more than one protein. This matrix of protein knots could then be purified by one of the methods mentioned above, bearing in mind that the high molecular weight of the structure makes it a good candidate for centrifugation techniques.
U slijedećem ostvarenju, predloženi izum može biti iskorišten za dodavanje cisteina proteinima. Često je poželjno uvesti cisteine u proteine kako bi se iskoristila jedinstvena reaktivnost cisteina za pričvršćivanje proteina za površine i za pričvršćivanje drugih molekula kao što su fluorofore za protein koji sadrži dodatni cistein. Mnogi proteini sadrže cisteine ili disulfide, otežavajući tako iskorištavanje cisteina koji je bio uveden u molekulu. Umetanjem triptofana u rep u blizinu cisteina koji je upotrijebljen za ukriženo vezanje čvora za cistein koji je uveden na mjesto proteina za modifikaciju, ova teškoća se može zaobići. Primjereno razlikama u apsorpciji spektra triptofana, tirozina, i fenilalanina, iridacija proteina sa zrakama od 295 nm će selektivno ciljati triptofanske ostatke. To će uzrokovati disrupciju obližnjeg disulfida, čineći reaktivnim tiolski ostatak na željenom cisteinu. Iridacija u prisutnosti grupe koja će biti dodana proteinu će omogućiti da protein bude etiketiran na željenom mjestu, čak i u proteina koji imaju nekoliko drugih disulfida. Dok je razumljivo da će drugi disulfidi u blizini triptofanskih ostataka također biti učinjeni reaktivnim, činjenica da većina proteina ima nekoliko triptofana znači da to obično ne predstavlja problem. In a further embodiment, the proposed invention can be used to add cysteine to proteins. It is often desirable to introduce cysteines into proteins to take advantage of the unique reactivity of cysteines to attach proteins to surfaces and to attach other molecules such as fluorophores to the protein containing the additional cysteine. Many proteins contain cysteines or disulfides, making it difficult to utilize the cysteine that was introduced into the molecule. By inserting a tryptophan into the tail near the cysteine used to cross-link the knot to the cysteine introduced into the protein site for modification, this difficulty can be circumvented. In accordance with the differences in absorption spectra of tryptophan, tyrosine, and phenylalanine, protein iridation with 295 nm rays will selectively target tryptophan residues. This will cause disruption of the nearby disulfide, making the thiol residue on the desired cysteine reactive. Iridation in the presence of a group to be added to the protein will allow the protein to be labeled at the desired site, even in proteins that have several other disulfides. While it is understandable that other disulfides near tryptophan residues will also be rendered reactive, the fact that most proteins have several tryptophans means that this is usually not a problem.
U dodatnom ostvarenju, predloženi izum može biti iskorišten da stvori i postigne visok prihod heterodimernih proteina. Pri stvaranju ekspresijske konstrukcije za proteinski čvor jedan dimer heterodimera može se sastojati od proteinske porcije. Ukoliko se u dimeru proteinske porcije obično ne pojavljuje porcija repa, porcija repa može biti fuzirana za dimer proteinske porcije. Čvor se može sastojati od drugog dimera heterodimera. Nakon ekspresije konstrukcije, postoji veća vjerojatnost da se heterodimer formira zahvaljujući porciji repa koja spaja dimere i sposobnosti formiranja disulfidne veze. Ova metoda zaobilazi probleme homodimerne formacije kada su dimeri heterodimera ko-eksprimirani u istoj stanici. In an additional embodiment, the present invention can be used to create and achieve a high yield of heterodimeric proteins. When creating an expression construct for a protein node, one dimer of a heterodimer can consist of a protein portion. If the tail portion usually does not appear in the protein portion dimer, the tail portion can be fused to the protein portion dimer. A knot may consist of another dimer of a heterodimer. Upon expression of the construct, a heterodimer is more likely to form due to the tail portion that joins the dimers and the ability to form a disulfide bond. This method circumvents the problems of homodimer formation when heterodimer dimers are co-expressed in the same cell.
Primjer 1 Example 1
Proteinski Čvorovi u studiji hCG – LHR Interakcije Protein Nodes in the study of hCG – LHR Interactions
Izvori hCG-a i antitijela upotrijebljenih u ovim studijama su bili opisani (Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem. J. 335, 611-617; Moyle, W. R.,Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031; Moyle, W. R., Matzuk, M. M., Campbell, R. K., Cogliani, E., Dean Emig, D. M., Krichevsky, A., Barnett, R. W. & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518). Konstrukcija sposobna za ekspresiju hCGβ-S138C je pripremljena kazetnom mutagenezom između prirodnog Apal mjesta na hCg cDNA i BamHI mjesta što je konstruirano nizvodno od terminacijskog kodona kao što je opisano (Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764; Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764.) Vektori upotrijebljeni za ekspresiju cisteinskih supstitucija α-podjedinice su također pripremljeni kao što je opisano (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry 2001). Konstrukcije što enkodiraju humanu α-podjedinicu ili cistein supstituirane analoge bile su ko-eksprimirane sa β-podjedinicom hCG-a ili hCGβ-S138C u stanicama COS-7 kao što je opisano (Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764). Materijali secernirani u kulturu medija su ispitivani sendvič imunoesejima (Moyle, W. R., Ehrlich, P. H. & Canfield, R. E. (1982) Proc. Natl. Acad. Sci. (USA) 79, 22245-2249) upotrebljavajući antitijelo A113 α-podjedinice za hvatanje i radioiodinirano antitijelo B110 β-podjedinice za detekciju. Oni su tretirani pri kiselom pH zbog promocije disocijacije heterodimera što im manjka disulfidna ukrižena veza, što je također opisano (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry 2001). Stanice kineskog hrčka (Chinese hamster cells: CHO stanice) koje previše eksprimiraju štakorski LHR upotrijebljene su za monitoriranje utjecaja analoga na sposobnost 125I-hCG-a da vežu vezajući LHR i izvabe akumulaciju cikličkog AMP-a kao što je prethodno izvješteno (Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem. J. 335, 611-617; Moyle, W. R., Matzuk, M. M., Campbell, R. K., Cogliani, E., Dean Emig, D. M., Krichevsky, A., Barnett, R. W. & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518; Moyle, W. R., Campbell, R. K., Myers, R. V., Bernard, M. P., Han, Y. & Wang, X. (1994) Nature 368, 251-255). The sources of hCG and antibodies used in these studies have been described (Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem. J. 335, 611-617; Moyle, W. R., Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y. & Wang, Y. (1995) J. Biol. Chem. 270, 20020-20031; Moyle, W. R., Matzuk, M. M., Campbell, R. K., Cogliani, E., Dean. Emig, D.M., Krichevsky, A., Barnett, R.W. & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518). A construct capable of expressing hCGβ-S138C was prepared by cassette mutagenesis between the native ApaI site on the hCg cDNA and a BamHI site constructed downstream of the termination codon as described (Campbell, R.K., Dean Emig, D.M. & Moyle, W.R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764; Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764.) Vectors used for the expression of α-subunit cysteine substitutions were also prepared as described (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs : implications for hormone folding and biological activity.Journal of Biological Chemistry 2001). Constructs encoding the human α-subunit or cysteine-substituted analogs were co-expressed with the β-subunit of hCG or hCGβ-S138C in COS-7 cells as described (Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. ( 1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764). Materials secreted into the culture medium were examined by sandwich immunoassays (Moyle, W. R., Ehrlich, P. H. & Canfield, R. E. (1982) Proc. Natl. Acad. Sci. (USA) 79, 22245-2249) using the A113 α-subunit antibody to capture and radioiodinated B110 β-subunit antibody for detection. They were treated at acidic pH to promote dissociation of heterodimers lacking a disulfide cross-link, which has also been described (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry 2001). Chinese hamster cells (CHO cells) overexpressing rat LHR were used to monitor the effect of analogs on the ability of 125 I-hCG to bind binding LHR and elicit cyclic AMP accumulation as previously reported (Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem. J. 335, 611-617; Moyle, W. R., Matzuk, M. M., Campbell, R. K., Cogliani, E., Dean Emig, D. M., Krichevsky, A., Barnett, R. W. & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518; Moyle, W. R., Campbell, R. K., Myers, R. V., Bernard, M. P., Han, Y. & Wang, X. (1994) Nature 368 , 251-255).
Alternativni pristup dešifriranju interakcija hCG-LHR uključuje identifikaciju površina hormona koje ostaju izložene u kompleksu hormon-receptor (Moyle, W. R., Matzuk, M. M., Campbell, R. K., Cogliani, E., Dean Emig, D. M., Krichevsky, A., Barnett, R. W. & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518). Kroz proces eliminacije, područja koja ostaju izložena u kompleksu hormon-receptor su tad mapirana na površini hormona da otkriju mjesta sposobna da kontaktiraju receptor. S obzirom da su ovi podaci skupljeni za vrijeme studija u kojima hormon zadržava svoju sposobnost interakcije s receptorom, podaci su puno lakše interpretirani od onih koji su ovisni o promjenama u interakcijama hormon-receptor. Većina metoda za detekciju ne-kontaktnih ostataka oslanja se na upotrebu testera monoklonih protutijela, proceduru koja je jako limitirana u rezoluciji. Kako bi se zaobišlo ovo ograničenje, mjerene su aktivnosti analoga u kojih je sigurnosni pojas zakvačen za α-podjedinicu... (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry. 2001.). Nekoliko ovih analoga ima u suštini istu aktivnost kao hCG, iako su imali dijelove sigurnosnog pojasa i karboksiterminal β-podjedinice pričvršćene za područja za koje se može očekivati da blokiraju interakciju hormon-receptor. Spajanje sigurnosnog pojasa sa α-podjedinicom ima potencijal da alterira konformaciju heterodimera, fenomen koji je možda odgovoran za nisku aktivnost nekih analoga. Nedavno, pronađeno je da je dugi nepravilni karboksiterminalni kraj β-podjedinice bio dovoljno pokretan da skenira mnogo površine heterodimera sve dok cistein uveden na ostatak 138 nije formirao disulfid s cisteinom uvedenim u α-podjedinicu. Sigurnosni pojasi ovih analoga su zakvačeni kako su u hCG-u, čineći manje vjerojatnim da mutacija alterira konformaciju heterodimera. Kao što je ovdje opisano, mnogi od ovih hCG analoga su mnogo aktivniji od onih u kojih je sigurnosni pojas pričvršćen za β-podjedinicu. Rezultati izvedenih studija također pokazuju da veliki dio petlje 2 α-podjedinice hCG-a, porcije hormona koja je vjerojatno u blizini receptorskog interface-a, ne kontaktira LHR.Uspoređene su aktivnosti hCG-analoga prije i poslije sputavanja porcije nepravilnog karboksiterminala β-podjedinice za cisteine uvedene u petlju 2 i karboksiterminala α-podjedinice disulfidom (vidi Slike 2 i 3). Izuzimajući analoge sa cisteinima na vršku petlje 2, heterodimeri kojima manjka ukrižena veza imaju bar 25% aktivnosti hCG-a u LHR vežućim i signalizirajućim analizama. Ovo sugerira da nekoliko ostataka u bilo kojoj regiji pridonosi više nego frakcija ukupne energije hCG-LHR vezanja i sukobljava se sa izvještajima da su karboksiterminalni ostaci esencijalni za efikasnost. Sputavanje β-podjediničnog karboksiterminalnog testera za površinu petlje 2 α-podjedinice okrenute petljama 1 i 3 β-podjedinice ima relativno malo utjecaja na vezanje ili signaliziranje, ukazujući na to da je mala vjerojatnost da ova porcija hormona kontaktira receptor. Gubitak aktivnosti uzrokovan spajanjem karboksiterminala β-podjedinice za druge ostatke može ukazivati na to da su ove porcije α-podjedinice blizu receptora. Aplikacija ove procedure za proučavanje interakcija hFSH sa FSH receptorom otkrila je da se hFSH veže za FSH receptore u sličnom sveukupnom stilu, ali različiti dijelovi hFSH čine ključne receptorske kontakte. Aplikacija ove procedure za proučavanje himernog hCG- hFSH liganda koji ima sposobnost interakcije i sa LH i sa FSH receptorima potvrdila je ove opservacije. Tako, nađeno je da su različite porcije himera bliže LHR nego FSHR . Slične strategije mutageneze bi trebale biti korisne za identificiranje površina drugih proteina koji ne sudjeluju u kontaktima protein-protein. An alternative approach to deciphering hCG-LHR interactions involves identifying hormone surfaces that remain exposed in the hormone-receptor complex (Moyle, W. R., Matzuk, M. M., Campbell, R. K., Cogliani, E., Dean Emig, D. M., Krichevsky, A., Barnett, R. W. & Boime, I. (1990) J. Biol. Chem. 265, 8511-8518). Through the process of elimination, the areas that remain exposed in the hormone-receptor complex are then mapped on the surface of the hormone to reveal sites capable of contacting the receptor. Since these data were collected during studies in which the hormone retains its ability to interact with the receptor, the data are much easier to interpret than those that depend on changes in hormone-receptor interactions. Most methods for non-contact residue detection rely on the use of monoclonal antibody testers, a procedure that is severely limited in resolution. To circumvent this limitation, the activities of analogs in which the seat belt is docked to the α-subunit were measured... (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard , M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry. 2001.). Several of these analogs have essentially the same activity as hCG, although they have portions of the seat belt and carboxyterminal β-subunit attached to regions that might be expected to block hormone-receptor interaction. Binding of the seat belt to the α-subunit has the potential to alter the conformation of the heterodimer, a phenomenon that may be responsible for the low activity of some analogs. Recently, it was found that the long irregular carboxy-terminal end of the β-subunit was mobile enough to scan much of the surface of the heterodimer until the cysteine introduced at residue 138 formed a disulfide with the cysteine introduced into the α-subunit. The seat belts of these analogs are pinned as they are in hCG, making it less likely that the mutation alters the conformation of the heterodimer. As described herein, many of these hCG analogs are much more active than those in which the seat belt is attached to the β-subunit. The results of the studies also show that a large part of loop 2 of the α-subunit of hCG, the portion of the hormone that is probably near the receptor interface, does not contact the LHR. cysteine introduced into loop 2 and the carboxy-terminal α-subunit by disulfide (see Figures 2 and 3). Excluding analogs with cysteines at the tip of loop 2, heterodimers lacking the cross-link have at least 25% hCG activity in LHR binding and signaling assays. This suggests that a few residues in any region contribute more than a fraction of the total energy of hCG-LHR binding and conflicts with reports that carboxyterminal residues are essential for efficiency. Restraint of the β-subunit carboxyterminal probe to the surface of loop 2 of the α-subunit facing loops 1 and 3 of the β-subunit has relatively little effect on binding or signaling, indicating that this portion of the hormone is unlikely to contact the receptor. Loss of activity caused by coupling of the carboxy-terminal β-subunit to other residues may indicate that these portions of the α-subunit are close to the receptor. Application of this procedure to study the interactions of hFSH with the FSH receptor revealed that hFSH binds to FSH receptors in a similar overall style, but different parts of hFSH make key receptor contacts. The application of this procedure to the study of a chimeric hCG-hFSH ligand capable of interacting with both LH and FSH receptors confirmed these observations. Thus, different portions of the chimera were found to be closer to LHR than to FSHR. Similar mutagenesis strategies should be useful for identifying surfaces of other proteins that do not participate in protein-protein contacts.
Ovdje opisane studije također ilustriraju aplikaciju ove procedure za identifikaciju mjesta za vezanje hFSH za FSH receptor i za usporedbu interakcija bifunkcionalnog hCG/hFSH analoga i sa LH i FSH receptorima. Analog β-podjedinice hFSH koji je enkodirao porciju ostataka karboksiterminala β-podjedinice hCG-a (fqdsssskapppslpspsrlpgpstdpilpg, SEQ ID NO:55) na njegovom β-podjediničnom karboksiterminalu je pripremljen s obzirom da hFSH nema «sigurnosni pojas» (porciju repa) (Slika 8, SEQ ID NO:41). Slično studijama izvedenim s hCG-om u kojeg je serinski ostatak 138 zamjenjen sa cisteinom, serinski ostatak 132 analoga β-podjedinice hFSH je zamjenjen cisteinom (Slika 8, SEQ ID NO:42). Analog S132C β-podjedinice hFSH je tada eksprimiran sa nekoliko α-podjediničnih analoga koji su sadržavali supstituirani cistein. Kao što je očekivano na osnovi iskustva sa sličnim analozima hCG-a, β-podjedinica hFSH analoga postala je ukriženo vezana sa α-podjedinicom preko disulfida kao što je dokazano činjenicom da je heterodimer bio puno stabilniji od nativnog hFSH pri niskom pH. Mnogi od ovih analoga su imali visoke aktivnosti u hFSH receptorskim analizama (vidi Sliku 13). Nekoliko razlika je detektirano u aktivnostima FSH-deriviranih analoga u usporedbi sa hCG-deriviranim analozima. Ove razlike otkrile su da je FSH interreagirao sa FSH-receptorom različito od interakcije hCG-a sa LH-receptorom na taj način omogućujući konstrukciju modela svakog kompleksa hormon-receptora. Rezultati također ukazuju na to da karboksiterminalna sekvenca β-podjedinice hCG-a može biti iskorištena kao «porcija repa» za dodavanje čvora na površinu proteina kojemu manjka prikladno mjesto ili «porcija repa» za činjenje toga. Netko iskusan u struci će prepoznati da mnoge druge sekvence osim karboksiterminala β-podjedinice hCG-a mogu biti iskorištene za ispunjavanje istog zadatka. Sekvenca «porcije repa» zahtijeva i da sekvenca bude dostatno duga toliko da cistein u porciji repa može dosegnuti cistein na površini proteina za kojeg će biti pričvršćen, i da sekvenca ne sadrži ostatke koji je sprječavaju da dosegne cistein na površini proteina. Ostaci koji bi spriječili cistein na porciji repa da dosegne cistein na površini proteina uključuju ostatke koji bi uzrokovali da se sekvenca savije u zasebnu domenu koja bi sekvestrirala cistein na porciji repa, ostatke kao što su oni koji sadrže signal za protein da postane pričvršćen za celularnu membranu tako sekvestrirajući cistein, ostatke koji imaju signal za protein da veže drugi protein tako sekvestrirajući cistein, ili ostatke koji su visoko nabijeni tako da blokiraju interakcije između površine proteina. The studies described here also illustrate the application of this procedure to identify the binding site of hFSH to the FSH receptor and to compare the interactions of a bifunctional hCG/hFSH analog with both the LH and FSH receptors. An hFSH β-subunit analog that encoded a portion of the carboxy-terminal residues of the hCG β-subunit (fqdssskapppslpspsrlpgpstdpilpg, SEQ ID NO:55) at its β-subunit carboxy-terminal was prepared given that hFSH does not have a "safety band" (tail portion) (Figure 8 , SEQ ID NO:41). Similar to the studies performed with hCG in which serine residue 138 was replaced with cysteine, serine residue 132 of the hFSH β-subunit analog was replaced with cysteine (Figure 8, SEQ ID NO:42). The S132C hFSH β-subunit analog was then co-expressed with several α-subunit analogs that contained a substituted cysteine. As expected based on experience with similar hCG analogs, the β-subunit of the hFSH analog became cross-linked to the α-subunit via a disulfide as evidenced by the fact that the heterodimer was much more stable than native hFSH at low pH. Many of these analogs had high activities in hFSH receptor assays (see Figure 13). Several differences were detected in the activities of FSH-derived analogs compared to hCG-derived analogs. These differences revealed that FSH interacted with the FSH-receptor differently from the interaction of hCG with the LH-receptor, thus allowing the construction of a model of each hormone-receptor complex. The results also indicate that the carboxy-terminal sequence of the β-subunit of hCG can be used as a "tail portion" to add a knot to the surface of a protein that lacks a suitable site or "tail portion" to do so. One skilled in the art will recognize that many other sequences besides the carboxy-terminal β-subunit of hCG can be used to accomplish the same task. The "tail portion" sequence also requires that the sequence be long enough that the cysteine in the tail portion can reach the cysteine on the surface of the protein to which it will be attached, and that the sequence does not contain residues that prevent it from reaching the cysteine on the protein surface. Residues that would prevent a tail cysteine from reaching a protein surface cysteine include residues that would cause the sequence to fold into a separate domain that would sequester the tail cysteine, residues such as those that contain a signal for the protein to become attached to the cell membrane thus sequestering a cysteine, residues that have a signal for a protein to bind another protein thus sequestering a cysteine, or residues that are highly charged so as to block protein-surface interactions.
Bifunkcionalni analozi glikoproteinskih hormona mogu se pripremiti međusobnom izmjenom dijelova njihovih sigurnosnih pojasa (U.S. Patent No. 5,508,261, Moyle et al). Za daljnje razabiranje razlika u interakcijama lutropina, kao što su hCG sa LH receptorom, i folitropina, kao što su hFSH sa FSH receptorom, pripremljen je analog hCG-a za kojeg je znano da veže i LH i FSH receptore. Ostaci 101-114 β-podjedinice hCG-a su zamijenjeni sa svojim nadopunama s β-podjedinice hFSH, poglavito su to ostaci 95-108 β-podjedinice hFSH (Slika 8, SEQ ID NO:38). Kao što je ranije učinjeno u slučaju hCG i hFSH analoga, serinski ostatak 138 u karboksiterminalu β-podjedinice ovog analoga zamijenjen je cisteinom (Slika 8, SEQ ID NO:39). Konstrukcija je eksprimirana u COS-7 stanicama s nekoliko α-podjediničnih analoga koji imaju supstituirani cistein. Heterodimeri koji su producirani u COS-7 stanicama su izmjereni upotrebom sendvič imunoanalize upotrebljavajući antitijela prema α- i β-podjedinicama hCG-a. Za mnoge je nađeno da su ukriženo vezani pomoću disulfida između α- i β-podjedinica na osnovi njihove povećane stabilnosti pri niskom pH. Neki od ovih analoga imali su signifikantno različite interakcije s LH i FSH receptorima. Na primjer, analog u kojeg je karboksiterminal β-podjedinice ukriženo vezan za cisteinski ostatak 37 α-podjedinice, dobro je interreagirao sa LH receptorima, i slabo sa FSH receptorima (vidi Slike 12-14). Ovo je potvrdilo otkriće da se interakcije hCG-a sa LH receptorima signifikantno razlikuju od onih hFSH sa FSH receptorima i osiguralo značajnu daljnju podršku za modele prema kojima svaki od ovih hormona interreagira sa svojim receptorima. Bifunctional analogues of glycoprotein hormones can be prepared by interchanging parts of their safety belts (U.S. Patent No. 5,508,261, Moyle et al). To further understand the differences in the interactions of lutropin, such as hCG with the LH receptor, and follitropin, such as hFSH with the FSH receptor, an hCG analog known to bind both LH and FSH receptors was prepared. Residues 101-114 of the β-subunit of hCG are replaced with their complements from the β-subunit of hFSH, mainly residues 95-108 of the β-subunit of hFSH (Figure 8, SEQ ID NO:38). As previously done in the case of hCG and hFSH analogs, serine residue 138 in the carboxy-terminal β-subunit of this analog was replaced with cysteine (Figure 8, SEQ ID NO:39). The construct was expressed in COS-7 cells with several α-subunit analogs having a substituted cysteine. Heterodimers produced in COS-7 cells were measured using a sandwich immunoassay using antibodies against the α- and β-subunits of hCG. Many are found to be disulfide cross-linked between the α- and β-subunits based on their increased stability at low pH. Some of these analogs had significantly different interactions with LH and FSH receptors. For example, an analog in which the carboxy-terminus of the β-subunit was cross-linked to cysteine residue 37 of the α-subunit interacted well with LH receptors, and weakly with FSH receptors (see Figures 12-14). This confirmed the finding that the interactions of hCG with LH receptors are significantly different from those of hFSH with FSH receptors and provided significant further support for models in which each of these hormones interacts with its receptors.
Rezultati the results
hCG α-podjedinični analozi i ili nativna β-podjedinica hCG-a ili hCGβ-S138C hCG α-subunit analogs and either the native β-subunit of hCG or hCGβ-S138C
COS-7 stanice koje su ko-transfekirane s vektorima što enkodiraju većinu α-podjediničnih analoga i ili nativnu β-podjedinicu (Tablica 1) ili hCGβ-S138C (Tablica 2) su sposobne za sakupljanje heterodimera i njihovo secerniranje u kulturu medija. Slabo ili nikako secernirani heterodimeri uključuju one koji imaju analoge β-podjedinice i α-podjedinice u kojih je cistein supstituiran umjesto ostataka Tyr37, Pro40, Asn52, i Y89C (Tablica 1). Slaba sekrecija αN52C/β može reflektirati odsustvo N-spojenog glikolizacijskog signala koji se normalno nalazi na ovoj poziciji hCG-a a potreban je za učinkoviu sekreciju heterodimera (Matzuk, M. M. & Boime, I. (1998) J. Cell. Biol. 106, 1049-1059). COS-7 cells co-transfected with vectors encoding most α-subunit analogs and either the native β-subunit (Table 1) or hCGβ-S138C (Table 2) are capable of accumulating heterodimers and secreting them into the culture medium. Weakly or not at all secreted heterodimers include those having β-subunit and α-subunit analogs in which cysteine is substituted for residues Tyr37, Pro40, Asn52, and Y89C (Table 1). The poor secretion of αN52C/β may reflect the absence of the N-linked glycolysis signal normally found at this position of hCG and required for efficient secretion of the heterodimer (Matzuk, M. M. & Boime, I. (1998) J. Cell. Biol. 106, 1049-1059).
Tablica 1. Produkcija heterodimera od strane COS-7 stanica, transfektiranim sa indiciranim podjediničnim konstrukcijama Table 1. Heterodimer production by COS-7 cells transfected with the indicated subunit constructs
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Većina ispitivanih α-podjediničnih analoga je detektirana u heterodimerima koji sadrže hCGβ-S138C (Tablica 2). hCGβ-S138C je spasila formaciju nekih analoga koji su slabo secernirani kad su eksprimirani s nativnom β-podjedinicom, uključujući Tyr37, Pro40, i Asn52 ali ne i Tyr89 (Tablica 2, Slika 1). Pokazalo se da mnogi od ovih heterodimera iz Tablice 2 imaju interpodjediničnu ukriženu vezu jer su bili lako detektirani nakon kratkog tretmana pri niskom pH. Heterodimeri koji imaju nativnu α-podjedinicu ili αG22C i αV53C α-podjedinične analoge su bili uništeni pri niskom pH sugerirajući da im manjka interpodjedinični disulfid. Međutim, samo frakcija heterodimera koja sadrži αQ5C, αQ27C, αP40C, αK51C, αL41C, αM71C, i αV76C α-podjedinične analoge se pokazala da je stabilizirana pomoću interpojediničnih disulfida (Tablica 2). Cisteini u α-podjediničnih analoga u analoga koji formiraju malo ili nimalo ukriženo vezanih heterodimera su na interface-u podjedinice ili su daleko od ostatka Asp111 β-podjedinice, prvog ostatka u karboksiterminalnoj ekstenziji β-podjedinice. Kao posljedica toga, pokazalo se da je cistein na β-podjediničnom ostatku 138 spriječen u dosezanju ovih α-podjediničnih cisteina. Ovaj fenomen sugerira da je većina interpodjediničnih disulfidnih ukriženih veza formirana nakon što su se podjedinice sakupile u heterodimer strukture slične hCG-u. Jedini ispitivani α-podjedinični analog koji nije uspio formirati heterodimer sa bilo β-podjedinicom hCG-a ili hCGβ-S138C imao je cistein na mjestu Tyr89. Dok ovaj tirozin nije esencijalan za savijanje α-podjedinice zato što može biti izbrisan ili promijenjen u druge ostatke osim cisteina bez disrupcije heterodimerne formacije (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495), zamjenjivanje tirozina s cisteinom moglo je uzrokovati disrupciju savijanja α-podjedinice (Chen, F., Wang, Y. & Puett, D. (1992) Mol. Endocrinol. 6, 914-919). Most of the tested α-subunit analogues were detected in heterodimers containing hCGβ-S138C (Table 2). hCGβ-S138C rescued the formation of some analogs that were poorly secreted when expressed with the native β-subunit, including Tyr37, Pro40, and Asn52 but not Tyr89 (Table 2, Figure 1). Many of these heterodimers in Table 2 were shown to have an intersubunit cross-link because they were readily detected after a short treatment at low pH. Heterodimers having the native α-subunit or the αG22C and αV53C α-subunit analogs were destroyed at low pH suggesting that they lack an intersubunit disulfide. However, only the heterodimer fraction containing the αQ5C, αQ27C, αP40C, αK51C, αL41C, αM71C, and αV76C α-subunit analogs was shown to be stabilized by intersubunit disulfides (Table 2). Cysteines in α-subunit analogs in analogs that form little or no cross-linked heterodimers are at the subunit interface or are far from the Asp111 β-subunit residue, the first residue in the carboxyterminal extension of the β-subunit. As a consequence, the cysteine at β-subunit residue 138 was shown to be prevented from reaching these α-subunit cysteines. This phenomenon suggests that most of the intersubunit disulfide cross-links are formed after the subunits have assembled into hCG-like heterodimer structures. The only α-subunit analog tested that failed to form a heterodimer with either the β-subunit of hCG or hCGβ-S138C had a cysteine at the Tyr89 position. While this tyrosine is not essential for α-subunit folding because it can be deleted or changed to residues other than cysteine without disrupting heterodimer formation (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495), replacing tyrosine with cysteine could cause disruption of α-subunit folding (Chen, F., Wang, Y. & Puett, D. (1992) Mol. Endocrinol. 6, 914-919).
Indicirane α-podjedinične konstrukcije i hCGβ-S138C Indicated α-subunit constructs and hCGβ-S138C
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a) Ova vrijednost izračunata je kao postotak materijala koji je ostao u uzorku nakon tretmana pri kiselom pH kao što je opisano u tekstu. a) This value was calculated as the percentage of material remaining in the sample after treatment at acidic pH as described in the text.
n) Ova vrijednost izračunata je kao omjer aktivnosti determinirane u sendvič esejima upotrebljavajući A111 i 125I-B111 u odnosu na one opservirane u A113 i 125I-B110 nakon tretmana pri niskom pH. Detektiranje imalo B111 vezanja indicira da je sigurnosni pojas zakvačen. Niske vrijednosti koje su opsevirane u nekim slučajevima mogu reflektirati sterični utjecaj β-podjediničnog karboksiterminala na sposobnost B111 da uđe u interakciju sa ukriženo vezanim heterodimerom. n) This value was calculated as the ratio of the activity determined in sandwich assays using A111 and 125I-B111 to that observed in A113 and 125I-B110 after treatment at low pH. Detecting any B111 buckles indicates that the seat belt is fastened. The low values observed in some cases may reflect the steric influence of the β-subunit carboxy terminal on the ability of B111 to interact with the cross-linked heterodimer.
Supstitucija cisteina za mnoge ostatke na petlji 2 α-podjedinice imala je mali utjecaj na receptor-vežuće signal-transdukcijske aktivnosti hCG-a (Tablica 3, Slika 2). Zamjenjivanje α-podjediničnih ostataka αMet47 (Tablica 3, Slika 2) i αLys51 (Einstein, M., Lin, W., Macdonald, G. J. & Moyle, W.R. (2001) Exp. Biol. Med. 226, 581-590) sa cisteinom reduciralo je aktivnosti heterodimera u analizama vezanja i signaliziranja u odnosu na one od hCG-a. Dok je analog u kojeg je αLys51 zamijenjen alaninom također imao značajno manju aktivnost od hCG-a (Einstein, M., Lin, W., Macdonald, G. J. & Moyle, W. R. (2001) Exp. Biol. Med. 226, 581-590), hCG-αM47A, analog u kojeg je αMet47 bio zamijenjen alaninom imao je približno jednaku aktivnost kao i hCG u obje analize (Tablica 3). Ovo sugerira da prisutnost metionina na α-podjediničnom ostatku 47 nije esencijalna za hCG aktivnost. Specifična uloga αLys51 u interakciji receptora ostaje da bude determinirana. Na osnovi otkrića da je heterodimer u kojeg je α-podjedinični ostatak 51 ukriženo vezan za β-podjedinični ostatak 99 pomoću disulfida aktivniji od onog u kojeg je αLys51zamijenjen cisteinom ili alaninom, pokazalo se da je vjerojatno da zamjenjivanje αLys51 postraničnog lanca može alterirati konformaciju heterodimera. Cysteine substitution for many residues in loop 2 of the α-subunit had little effect on the receptor-binding signal-transduction activities of hCG (Table 3, Figure 2). Replacement of α-subunit residues αMet47 (Table 3, Figure 2) and αLys51 (Einstein, M., Lin, W., Macdonald, G. J. & Moyle, W.R. (2001) Exp. Biol. Med. 226, 581-590) with cysteine. reduced heterodimer activities in binding and signaling assays compared to those of hCG. While an analogue in which αLys51 was replaced by alanine also had significantly less activity than hCG (Einstein, M., Lin, W., Macdonald, G. J. & Moyle, W. R. (2001) Exp. Biol. Med. 226, 581-590 ), hCG-αM47A, an analogue in which αMet47 was replaced by alanine had approximately the same activity as hCG in both assays (Table 3). This suggests that the presence of methionine at α-subunit residue 47 is not essential for hCG activity. The specific role of αLys51 in receptor interaction remains to be determined. Based on the discovery that a heterodimer in which α-subunit residue 51 is cross-linked to β-subunit residue 99 by a disulfide is more active than one in which αLys51 is replaced by cysteine or alanine, it has been shown that it is likely that substitution of the αLys51 side chain can alter the conformation of the heterodimer.
Tablica 3. Utjecaj mutacija u α-podjediničnoj petlji 2 i karboksiterminalu na heterodimeru lutropinske aktivnosti Table 3. Effect of mutations in the α-subunit loop 2 and the carboxy terminal on the heterodimer of lutropin activity
[image] [image]
a) Bazirano na koncentraciji analoga koji su determinirani sendvič imunoanalizom. Ove vrijednosti izračunate su iz IC50 vrijednosti iz rezimiranih eksperimenata. Nekoliko analoga nije testirano u ovim analizama zahvaljujući činjenici da su samo male količine producirane od strane transfektiranih COS-7 stanica. Kao što je također prikazano u Tablici 1, nismo pribavili nimalo stabilnih heterodimera slijedeći kiseli tretman α/hCgβ-S138C. a) Based on the concentration of analogs determined by sandwich immunoassay. These values were calculated from IC50 values from summarized experiments. Several analogs were not tested in these assays due to the fact that only small amounts were produced by transfected COS-7 cells. As also shown in Table 1, we did not obtain any stable heterodimers following acid treatment of α/hCgβ-S138C.
Izvješteno je da mijenjanje ostatka αLys44 petlje 2 α-podjedinice u alanin reducira hCG aktivnost za 100-pregiba ili više (Xia, H., Chen, F. & Puett, D. (1994) Endocrinol. 134, 1768-1770). Iz toga slijedi otkriće da je zamjena αLys44 i nekoliko obližnjih α-podjediničnih ostataka s cisteinom imala puno manji utjecaj na hCG aktivnost nego što je očekivano (Tablica 3). Za vrijeme dijela nepovezane studije za testiranje predviđanja učinjenih o naboju na vršku petlje 2 α-podjedinice na podjediničnoj kombinaciji (Slaughter, S., Wang, Y. H. , Myers, R. V. & Moyle, W. R. (1995) Mol. Cell. Endocrinol. 112, 21-25), hCG analog koji je imao glutamat i glutamin na mjestu lizina na ostacima 44 i 45, svaki ponaosob, je pripremljen (hCG-αK44E,K45Q) ,(Slika 15, SEQ ID NO 52). Neočekivano, ovaj analog je imao visoku aktivnost u oba analize kao i hCGα-K44A, ( Slika 15, SEQ ID NO 51) i hCGα-K44R, ( Slika 15, SEQ ID NO 53), analozi koji su imali alanin i arginin na mjestu αLys44 (Tablica 3, Slika 19). Visoka aktivnost potonjeg analoga očekivana je bazirajući se na činjenici da poput konja oblikovana α-podjedinica ima ovu istu supstituciju (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495; Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495). Ove studije su dosljedne u opservacijama učinjenim nakon zamjene ovih ostataka cisteinom. Ove opservacije su suprotne ranijim otkrićima (Xia, H., Chen, F. & Puett, D. (1994) Endocrinol. 134, 1768-1770), sugerirajući da niti jedan od visoko konzerviranih pozitivno nabijenih lizinskih ostataka u malom heliksu nađenom u petlji 2 α-podjedinice nije esencijalan za LHR interakcije. Alteration of the αLys44 residue of loop 2 of the α-subunit to alanine has been reported to reduce hCG activity by 100-fold or more (Xia, H., Chen, F. & Puett, D. (1994) Endocrinol. 134, 1768-1770). This resulted in the finding that replacement of αLys44 and several nearby α-subunit residues with cysteine had much less effect on hCG activity than expected (Table 3). During part of an unrelated study to test predictions made about the charge on the tip of loop 2 of the α-subunit on a subunit combination (Slaughter, S., Wang, Y. H. , Myers, R. V. & Moyle, W. R. (1995) Mol. Cell. Endocrinol. 112, 21 -25), an hCG analog that had glutamate and glutamine in place of lysine at residues 44 and 45, each individually, was prepared (hCG-αK44E,K45Q), (Figure 15, SEQ ID NO 52). Unexpectedly, this analog had high activity in both assays as did hCGα-K44A, (Figure 15, SEQ ID NO 51) and hCGα-K44R, (Figure 15, SEQ ID NO 53), analogs having alanine and arginine in the αLys44 (Table 3, Figure 19). The high activity of the latter analog is expected based on the fact that the horse-shaped α-subunit has this same substitution (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495; Pierce, J. G. & Parsons, TF (1981) Annu. Rev. Biochem. 50, 465-495). These studies are consistent with the observations made after replacing these residues with cysteine. These observations are contrary to earlier findings (Xia, H., Chen, F. & Puett, D. (1994) Endocrinol. 134, 1768-1770), suggesting that none of the highly conserved positively charged lysine residues in the small helix found in the loop 2 α-subunit is not essential for LHR interactions.
Eliminacija βCys26 Elimination of βCys26
Nađeno je da βCys110, cistein koji kvači karboksiterminalni kraj sigurnosnog pojasa za jezgru β-podjedinice, može postati ukriženo vezan za cistein koji je uveden u mnoge od ovih α-podjediničnih analoga ako je βCys110 spriječen u formiranju disulfida s β-podjediničnim ostatkom 26 (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry 50 , 46953-46960, 2001). Ovo otkriće promijenilo je poziciju sigurnosnog pojasa i eliminiralo epitop za antitijelo B111. Kako bi saznali da li su sigurnosni pojasi ovih ukriženih analoga bili pričvršćeni za βCys26, bile su uspoređene njihove sposobnosti da budu prepoznati od strane antitijela B111 i B110, antitijela što prepoznaje različit β-podjedinični epitop. Kao što je pokazano u Tablici 2, svaki ukriženo vezani heterodimer je bio prepoznat od strane B111, premda ne uvijek lako kao od strane B110, opservacija koja je sugerirala da je sigurnosni pojas bio zakvačen za βCys26 na isti način kao što je u hCG-u. Iako ne možemo isključiti mogućnost da je βCys110 zakvačen za cistein koji je bio umetnut u α-podjedinicu nekih analoga, ovo se čini jako nevjerojatno iz dvaju razloga. Prvo, sve β-podjedinice korištene u ovim studijama imaju cistein na ostatku 26. Eliminacija ovog cisteina na ostatku 26 je potrebna kako bi uzrokovala ukriženo vezanje sigurnosnog pojasa za cistein uveden u α-podjedinicu bez obzira na njihovu lokaciju (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry, 2001). I drugo, lokacija α-podjediničnog cisteina u ukriženih analoga što su prepoznati najmanje od strane B111 ( αY37C, αP40C, αL41C, αR42C, i αT86C ) je najbliža mjestu vezanja B111 (vidi Sliku 1). Ovo sugerira da je ukrižena veza možda stabilizirala poziciju β-podjediničnog karboksiterminala ovih analoga u poziciji koja je interferirala s pristupom B111 ka heterodimeru. It was found that βCys110, the cysteine that anchors the carboxy-terminal end of the β-subunit core seat belt, can become cross-linked to the cysteine introduced into many of these α-subunit analogs if βCys110 is prevented from forming a disulfide with the β-subunit residue 26 (Xing , Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry 50 , 46953 -46960, 2001). This discovery changed the position of the seat belt and eliminated the epitope for the B111 antibody. To find out whether the safety bands of these cross-linked analogs were attached to βCys26, their abilities to be recognized by antibodies B111 and B110, antibodies that recognize a different β-subunit epitope, were compared. As shown in Table 2, each cross-linked heterodimer was recognized by B111, although not always as readily as by B110, an observation that suggested that the seat belt was docked to βCys26 in the same way as in hCG . Although we cannot rule out the possibility that βCys110 is docked to a cysteine that was inserted into the α-subunit of some analogs, this seems highly unlikely for two reasons. First, all β-subunits used in these studies have a cysteine at residue 26. Elimination of this cysteine at residue 26 is required to cause cross-binding of the seat belt to the cysteine introduced into the α-subunit regardless of their location (Xing, Y., Lin , W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry, 2001). And second, the location of the α-subunit cysteine in the cross-linked analogs recognized least by B111 (αY37C, αP40C, αL41C, αR42C, and αT86C) is closest to the B111 binding site (see Figure 1). This suggests that the crosslink may have stabilized the β-subunit carboxyterminal position of these analogs in a position that interfered with access of B111 to the heterodimer.
Karboksiterminalni kraj α-podjedinice The carboxy-terminal end of the α-subunit
Smatralo se je da je karboksiterminalni kraj α-podjedinice esencijalan za LHR interakcije (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495; Yen, S. S. C., Llerena, O., Little, B. & Pearson, O. H. (1968) J. Clin. Endocrinol. Metabol. 28, 1763-1767). Prisustvo cisteina na karboksiterminalnom kraju α-podjedinice također je dovelo do male redukcije u aktivnosti heterodimera (Tablica 3, Slika 3), fenomen dosljedan s navodnom ulogom ove porcije hormona kao receptorskog kontakta (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495; Chen, F., Wang, Y. & Puett, D. (1992) Mol. Endocrinol. 6, 914-919). Usprkos tomu, ovi analozi su bili mnogo aktivniji nego analog kojemu manjka pet karboksiterminalnih α-podjediničnih ostataka (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495), što indicira da nijedan samostalan ostatak u ovoj regiji nije esencijalan za hormonsku aktivnost i da svaki čini tek mali doprinos ukupnoj energiji vezanja hCG-a za LHR. Bilo je začuđujuće pronaći da je heterodimer koji ima αK91C zadržao mnogo od hCG-ove efikasnosti (vidi Sliku 3), s obzirom da je izviješteno da αLys91 ima esencijalnu ulogu u signalnoj transdukciji (Yoo, J., Ji, I. & Ji, T. H. (1991) J. Biol. Chem. 266, 17741-17743) zahvaljujući navodnom kontaktu što ga ostvaruje sa ostatkom aspartne kiseline u transmembranskoj domeni (Ji, I., Zeng, H. & Ji, T. H. (1993) J. Biol. Chem. 268, 22971-22974). Otkriće da je hCG-αK91C zadržao značajnu efikasnost je nedosljedno s ovim prijedlogom. Kao rezultat, pripremljeni su hCG-αK91E i hCG-αK91M; ispitane su njihove aktivnosti. Izviješteno je da ovi analozi imaju vrlo nisku efikasnost (Yoo, J., Ji, I. & i, T. H. (1991) J. Biol. Chem. 266, 17741-17743). Kao što je prikazano na Slici 17, oba analoga su imala značajnu efikasnost, iako je analog koji sadržava glutamat interreagirao s LHR aproksimativno 10-pregiba nižim afinitetom nego hCG. Ova opservacija je dosljedna s opservacijama učinjenim kada je αLys91 konvertiran u cistein (Tablica 3, Slika 3) i može ukazivati da ovaj lizinski ostatak nije bitan za signalnu transdukciju kao što je prethodno izviješteno (Yoo, J., Ji, I. & i, T.H. (1991) J. Biol. Chem. 266, 17741-17743). The carboxy-terminal end of the α-subunit was thought to be essential for LHR interactions (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495; Yen, S. S. C., Llerena, O., Little, B. & Pearson, O. H. (1968) J. Clin. Endocrinol. Metabol. 28, 1763-1767). The presence of a cysteine at the carboxy-terminal end of the α-subunit also led to a small reduction in heterodimer activity (Table 3, Figure 3), a phenomenon consistent with the putative role of this portion of the hormone as a receptor contact (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev . Biochem. 50, 465-495; Chen, F., Wang, Y. & Puett, D. (1992) Mol. Endocrinol. 6, 914-919). Nevertheless, these analogs were much more active than an analog lacking the five carboxy-terminal α-subunit residues (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495), indicating that no single residue in this region is not essential for hormonal activity and that each makes only a small contribution to the total binding energy of hCG to LHR. It was surprising to find that the heterodimer bearing αK91C retained much of hCG's efficacy (see Figure 3), given that αLys91 has been reported to play an essential role in signal transduction (Yoo, J., Ji, I. & Ji, T. H. (1991) J. Biol. Chem. 266, 17741-17743) due to its putative contact with an aspartic acid residue in the transmembrane domain (Ji, I., Zeng, H. & Ji, T. H. (1993) J. Biol. Chem. 268, 22971-22974). The finding that hCG-αK91C retained significant efficacy is inconsistent with this proposal. As a result, hCG-αK91E and hCG-αK91M were prepared; their activities were examined. These analogs have been reported to have very low efficacy (Yoo, J., Ji, I. & i, T. H. (1991) J. Biol. Chem. 266, 17741-17743). As shown in Figure 17, both analogs had significant efficacy, although the glutamate-containing analog interacted with LHR with approximately 10-fold lower affinity than hCG. This observation is consistent with observations made when αLys91 is converted to cysteine (Table 3, Figure 3) and may indicate that this lysine residue is not essential for signal transduction as previously reported (Yoo, J., Ji, I. & i, T. H. (1991) J. Biol. Chem. 266, 17741-17743).
Karboksiterminal β-podjedinice i α-podjedinična petlja 2 Carboxyterminal β-subunit and α-subunit loop 2
Mnogi od acido-stabilnih analoga u kojih je karboksiterminal β-podjedinice bio ukriženo vezan za ostatke petlje 2 α-podjedinice su imali značajne aktivnosti u receptor-vežućim i signal-transdukcijskim analizama (Tablica 3, Slika 4). Oni uključuju one u kojih je pričvršćen za ostatke 35, 37, 40, 44, 42, 45, 50, i 52 (Tablica 3). Pričvršćivanje karboksiterminala β-podjedinice za ostatak 47 petlje 2 α-podjedinice je zamalo poništilo receptor-vežuću aktivnost i sparivajući ga s ostatkom 43 i 46 reduciralo aktivnost heterodimera za polovicu. Postranični lanci ostataka petlje 2 α-podjedinice se u gotovo svih najviše aktivnih analoga projiciraju prema β-podjediničnim petljama 1 i 3. Ovo sugerira da površina petlje 2 α-podjedinice što je okrenuta β-podjediničnim petljama 1 i 3 ne kontaktira LHR. Postranični lanci α-podjediničnih ostataka 43 i 46 projiciraju se prema sigurnosnom pojasu i onaj od ostatka 47 okrenut je prema maloj petlji sigurnosnog pojasa formiranoj disulfidom između Cys93 i Cys100. Zbog toga, gubitak u aktivnosti uzrokovan spajanjem karboksiterminalne porcije za ova mjesta može izazvati disrupciju u interakciji između ovih porcija α-podjedinične petlje 2 s receptorom ili alterirati konformaciju petlje 2 α-podjedinice na način da reducira sposobnost hormona da intereagira s receptorom. Many of the acid-stable analogs in which the carboxy-terminus of the β-subunit was cross-linked to loop 2 residues of the α-subunit had significant activities in receptor-binding and signal-transduction assays (Table 3, Figure 4). These include those in which it is attached to residues 35, 37, 40, 44, 42, 45, 50, and 52 (Table 3). Attaching the β-subunit carboxy-terminal to residue 47 of loop 2 of the α-subunit nearly abrogated the receptor-binding activity and pairing it with residues 43 and 46 reduced the activity of the heterodimer by half. The side chains of α-subunit loop 2 residues in almost all of the most active analogs project toward β-subunit loops 1 and 3. This suggests that the surface of α-subunit loop 2 facing β-subunit loops 1 and 3 does not contact LHR. The side chains of α-subunit residues 43 and 46 project toward the seat belt and that of residue 47 faces the small loop of the seat belt formed by the disulfide between Cys93 and Cys100. Therefore, loss of activity caused by joining the carboxy-terminal portion to these sites may disrupt the interaction between these portions of α-subunit loop 2 with the receptor or alter the conformation of loop 2 of the α-subunit in such a way as to reduce the ability of the hormone to interact with the receptor.
Dužina β-podjediničnog karboksiterminala sugerira da ostatak 138 može biti pričvršćen za ostatke α-podjedinične petlje 2 prelazeći preko β-podjediničnih petlji 1 i 3 (vidi Sliku 1). Odatle, karboksiterminalna porcija β-podjedinice možda ne zauzima prostor u brazdi između α-podjedinične petlje 2 i β-podjediničnih petlji 1 i 3 u bilo kojeg od ovih analoga. Za proučavanje aktivnosti analoga u kojih ova porcija karboksiterminalne porcije β-podjedinice zauzima ovu brazdu, pripremljeni su analozi kojima manjkaju β-podjedinični ostaci 116-135 i 121-135. U ovih analoga β-karboksiterminal je bio prekratak da formira ukriženu vezu sa α-podjediničnim ostacima u petlji 2 bez prolaženja kroz ovu interpodjediničnu brazdu. Ovi analozi su imali značajne aktivnosti u LHR vežućim i signalizacijskim analizama (vidi Sliku 4), osiguravajući dodatnu podršku za koncept da ova porcija α-podjedinične petlje 2 ne kontaktira LHR. The length of the β-subunit carboxyterminus suggests that residue 138 may be attached to α-subunit loop 2 residues crossing over β-subunit loops 1 and 3 (see Figure 1 ). Hence, the carboxy-terminal portion of the β-subunit may not occupy the space in the groove between α-subunit loop 2 and β-subunit loops 1 and 3 in either of these analogs. To study the activity of analogs in which this portion of the carboxyterminal portion of the β-subunit occupies this groove, analogs lacking β-subunit residues 116-135 and 121-135 were prepared. In these analogs, the β-carboxyterminal was too short to form a cross-link with the α-subunit residues in loop 2 without passing through this intersubunit groove. These analogs had significant activities in LHR binding and signaling assays (see Figure 4 ), providing further support for the concept that this portion of α-subunit loop 2 does not contact the LHR.
β-podjedinica i α-podjedinične petlje 1 i 3 β-subunit and α-subunit loops 1 and 3
Kako bi saznali da li β-podjedinični ostatak 138 može postati ukriženo vezan za druge porcije α-podjedinične jezgre, eksprimiran je hCG-β138C sa nekoliko α-podjediničnih analoga što sadrže cistein na mjestu ostatka u petljama 1 i 3. S izuzetkom heterodimera koji je imao cistein na mjestu αSer64, samo male količine rezultirajućih heterodimera su bile ukriženo vezane što je otkriveno pomoću njihove niske stabilnosti pri kiselom pH (Tablica 2). Cisteini mnogih α-podjediničnih analoga u ovih heterodimera su bili locirani mnogo dalje od karboksiterminalnog kraja sigunosnog pojasa nego oni u analoga št su sudjelovali u interpodjediničnim ukriženim vezama i, možda su bili izvan krajnjeg domašaja unutar kojeg bi bili efikasno dosegnuti od strane karboksiterminalne regije β-podjedinice (vidi Sliku 1). Heterodimer αS64C/hCG-β138C je imao značajnu aktivnost u receptor-vežućim i signalizacijskim analizama (Tablica 3), pokazujući da ovaj α-podjedinični ostatak ne stvara esencijalne kontakte s LHR. To find out whether β-subunit residue 138 can become cross-linked to other portions of the α-subunit core, hCG-β138C was expressed with several α-subunit analogs containing cysteine at the residue site in loops 1 and 3. With the exception of the heterodimer that had a cysteine at the αSer64 site, only small amounts of the resulting heterodimers were cross-linked as revealed by their low stability at acidic pH (Table 2). The cysteines of many of the α-subunit analogs in these heterodimers were located much further from the carboxyterminal end of the sigmoid band than those of the analogs involved in intersubunit cross-links and, perhaps, were beyond the ultimate reach within which they would be efficiently reached by the carboxyterminal region of β- subunits (see Figure 1). The αS64C/hCG-β138C heterodimer had significant activity in receptor-binding and signaling assays (Table 3), demonstrating that this α-subunit residue does not make essential contacts with the LHR.
Ukriženo vezanje β-podjediničnog karboksiterminala za ostatke u α-podjediničnom karboksiterminalu je imalo puno veći utjecaj na interakcije heterodimera sa LHR nego supstitucije cisteina u bilo kojoj podjedinici (vidi Sliku 5). Ovim je sugerirano da je α-podjedinični karboksiterminal blizu interface-a LH receptora kao što je predloženo bazirajući se na studijama izvedenim prije više od 25 godina (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465-495). Usprkos tomu, moguće je da pričvršćivanje karboksiterminalnog kraja β-podjedinice za ovu regiju alterira strukturu heterodimera ili uzrokuje da kraj β-podjedinice prođe blizu drugih porcija hormona koje kontaktiraju receptor. Cross-linking of the β-subunit carboxyterminal to residues in the α-subunit carboxyterminal had a much greater effect on the interactions of the heterodimer with LHR than cysteine substitutions in either subunit (see Figure 5). This suggests that the α-subunit carboxy terminus is close to the LH receptor interface as suggested based on studies performed more than 25 years ago (Pierce, J. G. & Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 465- 495). Nevertheless, it is possible that attachment of the carboxy-terminal end of the β-subunit to this region alters the structure of the heterodimer or causes the end of the β-subunit to pass close to other portions of the hormone that contact the receptor.
Proteinski čvorovi – hCG-β-laktamaza Protein knots – hCG-β-lactamase
Ove studije osiguravaju značajnu podršku ideji da većina ostataka u α-podjediničnoj petlji 2 ne sudjeluje u esencijalnim LH receptorskim kontaktima. Međutim, studije ne isključuju mogućnost da je ova porcija hormona u blizini receptorskog interface-a. Kako bi se latili ovog spornog pitanja pripremljeni su hCG β-podjedinični analozi u kojih je β-laktamaza, globularni protein po veličini sličan hCG-u, pričvršćena za specifična mjesta na α-podjedinici. Ovo je ostvareno fuzijom β-laktamaze za ostatke 140 i 145 hCG-βS138C što rezultira s hCG-βS138C-βLA140 i hCG-βS138C-LA145, odnoseći se na svaki ponaosob. Potonji analog imao je “razmak” od sedam ostataka između cisteina koji je trebao biti ukriženo vezan za α-podjedinicu i aminoterminala β-laktamaze. Ko-ekspresija ovih β-podjediničnih analoga s α-podjediničnim analozima αT46C, αL48C, αS64C, i αS92Cdovela je do formiranja acido-stabilnih heterodimera (vidi Sliku 6). Ovo je pokazalo da prisustvo β-laktamaze nije spriječilo β-podjedinični ostatak 138 da se pričvrsti za α-podjedinicu. These studies provide significant support for the idea that most residues in α-subunit loop 2 do not participate in essential LH receptor contacts. However, studies do not exclude the possibility that this portion of the hormone is near the receptor interface. To address this controversial issue, hCG β-subunit analogs were prepared in which β-lactamase, a globular protein similar in size to hCG, was attached to specific sites on the α-subunit. This was accomplished by β-lactamase fusion to residues 140 and 145 of hCG-βS138C resulting in hCG-βS138C-βLA140 and hCG-βS138C-LA145, respectively. The latter analog had a seven-residue “gap” between the cysteine that should be cross-linked to the α-subunit and the amino-terminus of the β-lactamase. Co-expression of these β-subunit analogs with the α-subunit analogs αT46C, αL48C, αS64C, and αS92C led to the formation of acid-stable heterodimers (see Figure 6). This indicated that the presence of β-lactamase did not prevent the β-subunit residue 138 from attaching to the α-subunit.
Ovi hCG-β-laktamaza analozi su imali puno nižu biološku aktivnost od korespondirajućih analoga kojima je manjkala β-laktamaza (vidi Sliku 6). These hCG-β-lactamase analogs had much lower biological activity than the corresponding β-lactamase-deficient analogs (see Figure 6).
Povezano sa rezultatima ranijih studija dizajniranih da identificiraju porcije α-podjedinice koje nisu uključene u ključne receptorske kontakte (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W. R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry. 2001.), ove opservacije proširuju sadašnje znanje o površinama hormona za koje se pokazalo da ne kontaktiraju LHR i sugeriraju da brazda između α-podjedinične petlje 2 i β-podjediničnih petlji 1 i 3 ne sudjeluje u esencijalnim LH receptorskim kontaktima, što je ključni zahtjev ranijeg modela (Moyle, W. R. , Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y. & Wang, Y. (1995) J, Biol. Chem. 270, 20020-20031). Uistinu, bazirano na sposobnostima monoklonalnih antitijela da prepoznaju hCG i hCG analoge vezane za LHR na površini stanica (Wang, Y. H., Bernard, M. P. & Moyle,W. R. (2000) Mol.Cell. Endocrinol. 170, 67-77) i sposobnosti hCG/hFSH i hCG/hTSH himera da vežu LHR (Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764; Campbell, R. K.,Bergert, E. R. , Wang, Y., Morris, J. C. & Moyle, W. R. (1997) Nature Biotech. 15, 439-443; Grossmann, M., Szkudlinski, M. W., Wong, R., Dias, J. A., Ji, T. H. & Weintraub, B. D. (1997) J. Biol. Chem. 272, 15532-15540; Moyle, W. R., Campbell, R. K., Myers, R. V., Bernard, M. P., Han, Y. & Wang,X. (1994) Nature 368 251-255), pokazalo se da nekoliko hCG-specifičnih ostataka sudjeluje u esencijalnim LHR kontaktima. Ostaci koji imaju najveći utjecaj na hCG aktivnost su locirani u sigurnosnom pojasu, ali čak i većina ovih može biti alterirana jedan-po-jedan bez disrupcije hormon-receptorskih interakcija za više od nekoliko pregiba (Han, Y., Bernard, M. P. & Moyle, W. R. (1996) Mol. Cell. Endocrinol. 124 , 151-161.) Bazirano na ovim opservacijama, otkriće da dva udaljena mjesta na receptoru, čini se, utječu na hCG-LRH interakcije (Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem. J. 335, 611-617), te otkriće da su interakcije lutropina sisavaca sa ljudskim LHR osobito osjetljive na male promjene u konformaciji hormona, predloženo je da interakcije glikoproteinskih hormona i njihovih receptora nisu dominirane s relativno malo kontakata, kao što su one nađene između hormona rasta i njegovog receptora (Clackson, T. & Wells, J. A. (1995) Science 267, 383-386; Wells, J. A. (1996) Proc. Natl. Acad. Sci. (USA) 83, 1-6.) Related to the results of earlier studies designed to identify portions of the α-subunit that are not involved in key receptor contacts (Xing, Y., Lin, W., Jiang, M., Myers, R. V., Cao, D., Bernard, M. P. & Moyle, W.R. Alternatively folded choriogonadotropin analogs: implications for hormone folding and biological activity. Journal of Biological Chemistry. 2001), these observations extend current knowledge of hormone surfaces that have been shown not to contact the LHR and suggest that the groove between α-subunit loop 2 and β-subunit loops 1 and 3 do not participate in essential LH receptor contacts, a key requirement of the earlier model (Moyle, W. R. , Campbell, R. K., Rao, S. N. V., Ayad, N. G., Bernard, M. P., Han, Y. & Wang, Y. (1995) J, Biol. Chem. 270, 20020-20031). Indeed, based on the ability of monoclonal antibodies to recognize hCG and hCG analogs bound to LHR on the cell surface (Wang, Y. H., Bernard, M. P. & Moyle, W. R. (2000) Mol.Cell. Endocrinol. 170, 67-77) and the ability of hCG/ hFSH and hCG/hTSH chimeras to bind LHR (Campbell, R. K., Dean Emig, D. M. & Moyle, W. R. (1991) Proc. Natl. Acad. Sci. (USA) 88, 760-764; Campbell, R. K., Bergert, E. R. , Wang, Y., Morris, J. C. & Moyle, W. R. (1997) Nature Biotech. 15, 439-443; Grossmann, M., Szkudlinski, M. W., Wong, R., Dias, J. A., Ji, T. H. & Weintraub, B. D. ( 1997) J. Biol. Chem. 272, 15532-15540; Moyle, W. R., Campbell, R. K., Myers, R. V., Bernard, M. P., Han, Y. & Wang, X. (1994) Nature 368 251-255), showed several hCG-specific residues are involved in essential LHR contacts. Residues that have the greatest impact on hCG activity are located in the safety band, but even most of these can be altered one-by-one without disrupting hormone-receptor interactions by more than a few folds (Han, Y., Bernard, M. P. & Moyle, W. R. (1996) Mol. Cell. Endocrinol. 124 , 151-161.) Based on these observations, the discovery that two distant sites on the receptor appear to influence hCG-LRH interactions (Bernard, M. P., Myers, R. V. & Moyle, W. R. (1998) Biochem. J. 335, 611-617), and the finding that interactions of mammalian lutropin with human LHR are particularly sensitive to small changes in hormone conformation, suggested that interactions between glycoprotein hormones and their receptors are not dominated by relatively few contacts, such as those found between growth hormone and its receptor (Clackson, T. & Wells, J. A. (1995) Science 267, 383-386; Wells, J. A. (1996) Proc. Natl. Acad. Sci. (USA) 83, 1 -6.)
Primjer 2 Example 2
Proteinski čvorovi za studiju sidrenja hFSH u FSH receptore Protein nodes for the study of hFSH anchoring in FSH receptors
cDNA kodirajuća sekvenca za hFSH β-podjedinicu je pribavljena od Christie Kelton, Ares Advanced Tehnology, odjel Serono, 280 Pond Street, Randolph MA koja odgovara translatiranoj porciji mRNA. Ilustrirana je aminokiselinska sekvenca ove β-podjedinice kojoj manjka vodeći peptid (Slika 8, SEQ ID NO: 40). hSFH cDNA je modificirana PCR-om i kazetnom mutagenezom za stvaranje konstrukcije nazvane FC1-108β (Slika 8, SEQ ID NO: 41), koja enkodira hSFH ostatke 1-108 i hCG ostatke 115-145 u tandemu. Aminokiselinska sekvenca od FC1-108β s manjkom vodećeg peptida pronađena u hSFH β-podjedinici je također ilustrirana na Slici 8. Analog od FC1-108β u kojeg je ostatak Ser132 konvertiran u Cys pripremljen je zamjenom Xhol-Apal fragmenta konstrukcije što enkodira hCG S138Cβ s onim od FC1-108β da se stvori FC1-108 S132Cβ (Slika 8, SEQ ID NO: 42). FC1-108, S132Cβ je eksprimiran s nekoliko α-podjediničnih analoga prikazanih na Slici 7 u COS-7 stranicama koristeći metode opisane u primjeru 1. Heterodimeri secernirani od stanica u kulturu medija su mjereni korištenjem antitijela A113 i 125I-B603. Prvospomenuti je antitijelo spram α-podjedinice, a drugi je monoklonalno antitijelo koje se veže za β-podjedinicu hSFH. Purificirani hSFH je upotrijebljen kao standard. Slijedio je kiseli tretman kako bi se disocirao bilo koji heterodimer koji nije ukriženo vezan, materijal je re-analiziran sa A113 i 125I-B603 kao gore da bi se determinirala količina ukriženo vezanog materijala u uzorku. Testirane su sposobnosti rezultirajućih ukriženih analoga da izvabe akumulaciju cikličkog AMP-a u CHO stanicama koje eksprimiraju FSH receptore. The cDNA coding sequence for the hFSH β-subunit was obtained from Christie Kelton, Ares Advanced Technology, Serono Division, 280 Pond Street, Randolph MA corresponding to the translated portion of the mRNA. The amino acid sequence of this β-subunit lacking the leader peptide is illustrated (Figure 8, SEQ ID NO: 40). The hSFH cDNA was modified by PCR and cassette mutagenesis to create a construct named FC1-108β (Figure 8, SEQ ID NO: 41), which encodes hSFH residues 1-108 and hCG residues 115-145 in tandem. The amino acid sequence of FC1-108β lacking the leader peptide found in the hSFH β-subunit is also illustrated in Figure 8. An analog of FC1-108β in which the Ser132 residue was converted to Cys was prepared by replacing the Xhol-Apal fragment of the construct encoding hCG S138Cβ with that of FC1-108β to generate FC1-108 S132Cβ (Figure 8, SEQ ID NO: 42). FC1-108, S132Cβ was expressed with several α-subunit analogs shown in Figure 7 in COS-7 cells using the methods described in Example 1. Heterodimers secreted from the cells into the culture medium were measured using antibodies A113 and 125I-B603. The first mentioned is an antibody against the α-subunit, and the second is a monoclonal antibody that binds to the β-subunit of hSFH. Purified hSFH was used as a standard. Following an acid treatment to dissociate any non-cross-linked heterodimer, the material was re-analyzed with A113 and 125 I-B603 as above to determine the amount of cross-linked material in the sample. The abilities of the resulting cross-linked analogs to elicit cyclic AMP accumulation in CHO cells expressing FSH receptors were tested.
Kao što je prikazano na Slici 11, utjecaj čvora na α-podjedinične ostatke 92, 64, 48, 46, 42, 90, 43, 88, i 86 rezultiralo je progresivnim gubitkom aktivnosti analoga. Ovo pokazuje da α-podjedinični ostatak 35 nije u blizini FSH receptorskog interface-a, te se čini da je nekoliko drugih ostataka u blizini receptorskog interface-a. Za razliku od interakcija hCG-a s LH-receptorom, prisutnost čvora na ostacima 42 i 43 je puno više bilo inhibitorno spram vezanja FSH analoga za njegov receptor. Ovo je otkrilo da je površina dijela α-podjedinične petlje 2 bila puno bliža FSH receptoru nego LH receptoru kada FSH i hCG interreagiraju svaki sa svojim receptorom. Otkriće da pričvršćivanje čvora na α-podjedinični ostatak 86 obaju receptora je pokazalo da je ova porcija obaju liganda blizu receptorskog interface-a. As shown in Figure 11, the impact of the knot on the α-subunit residues 92, 64, 48, 46, 42, 90, 43, 88, and 86 resulted in a progressive loss of activity of the analog. This indicates that the α-subunit residue 35 is not in the vicinity of the FSH receptor interface, and several other residues appear to be in the vicinity of the receptor interface. In contrast to the interactions of hCG with the LH-receptor, the presence of a knot at residues 42 and 43 was much more inhibitory to the binding of the FSH analogue to its receptor. This revealed that the surface area of the α-subunit loop 2 was much closer to the FSH receptor than to the LH receptor when FSH and hCG each interacted with their respective receptors. The finding that knot attachment at α-subunit residue 86 of both receptors indicated that this portion of both ligands is close to the receptor interface.
Primjer 3 Example 3
Proteinski čvorovi za studiju sidrenja bifunkcionalnog hCG/hSFH himera za LH I FSH receptore Protein nodes for the study of anchoring of the bifunctional hCG/hSFH chimera to LH and FSH receptors
cDNA sekvenca što enkodira himer u kojeg su hCG β-podjedinični kodoni za aminikiseline 110-114 zamijenjeni sa njihovim hFSH β-podjediničnim nadopunama, pripremljena je standardnim metodama. Aminokiselinska sekvenca ovog himera nazvana CFC101-114ß je prikazana na Slici 8 (SEQ ID NO:38) s nedostatkom vodećih ostataka. Analog od CFC101-114ß u kojeg je ostatak Ser138 konvertiran u Cys pripremljen je zamjenom Xhol-Apal fragmenta konstrukcije što enkodira hCG-S138ß s onim od CFC101-114ß da se stvori CFC101-114ß, S138C (Slika 8, SFQ ID NO:39). CFC101-114ß, S138C je komprimiran s nekoliko α-podjediničnih analoga prikazanih na Slici 7 u COS-7 stranicama koristeći metode prikazane u Primjeru 1. Heterodimeri koje su stranice secernirale u kulturu medija izmjerene su korištenjem antitijela A113 i 125I-B110. Prvonavedeni jest antitijelo na α-podjedinicu, a drugi je monoklonalno antitijelo što se veže za β-podjedinicu hCG-a. Purificirani hCG upotrijebljen je kao standard. Slijedio je kiseli tretman kako b disocirao bilo koji heterodimer koji nije ukriženo vezan, materijal je re-analiziran sa A113 i 125I-B110 kao gore, da bi se determinirala koncentracija ukrižano vezanog materijala u uzorku. Testirane su sposobnosti rezultirajućih ukriženo vezanih analoga da inhibiraju vezanje 125I-hCG za CHO stanice što eksprimiraju LH receptore i da inhibiraju vezanje 125I-hFSH za CHO stanice što eksprimiraju FSH receptore (vidi Sliku 12). Također su testirane njihove sposobnosti da izvabe akumulaciju cikličkog AMP-a u CHO stanicama koje eksprimiraju LH receptore i CHO stanicama što eksprimiraju FSH receptore. A cDNA sequence encoding a chimera in which the hCG β-subunit codons for amino acids 110-114 were replaced with their hFSH β-subunit complements was prepared by standard methods. The amino acid sequence of this chimera designated CFC101-114ß is shown in Figure 8 (SEQ ID NO:38) with leader residues missing. An analog of CFC101-114ß in which the Ser138 residue was converted to Cys was prepared by replacing the Xhol-Apal fragment of the construct encoding hCG-S138ß with that of CFC101-114ß to generate CFC101-114ß, S138C (Figure 8, SFQ ID NO:39) . CFC101-114ß, S138C was compressed with several α-subunit analogs shown in Figure 7 in COS-7 cells using the methods described in Example 1. Heterodimers secreted by the cells into the culture medium were measured using antibodies A113 and 125I-B110. The first is an antibody to the α-subunit, and the second is a monoclonal antibody that binds to the β-subunit of hCG. Purified hCG was used as a standard. Following an acid treatment to dissociate any non-cross-linked heterodimer, the material was re-analyzed with A113 and 125 I-B110 as above, to determine the concentration of cross-linked material in the sample. The abilities of the resulting cross-linked analogs to inhibit binding of 125 I-hCG to CHO cells expressing LH receptors and to inhibit binding of 125 I-hFSH to CHO cells expressing FSH receptors were tested (see Figure 12). Their abilities to induce accumulation of cyclic AMP in CHO cells expressing LH receptors and CHO cells expressing FSH receptors were also tested.
Kao što je prikazano na Slici 12 prisutnost čvora na α-podjediničnom ostatku 35 nije interferirala sa sposobnošću himera da blokira vezanje radiodiniranog hCG-a ili radiodiniranog FSH za LH ili FSH receptore, odnoseći se na svakoga pojedinog prikladnog bifunkcionalnog himera kojemu manjka većina karboksiterminala β-podjedinice hCG-a. Međutim, prisustvo čvora na α-podjediničnom ostatku 37 dovelo je do dramatično različitog rezultata. Dakle, ovaj je čvor imao vrlo malo utjecaja na sposobnost himera da se veže na LH receptore ali je gotovo eliminirao njegovu sposobnost da se veže za FSH receptore. Prisutnost čvora na α-podjediničnim ostacima 43 i 46 reducirala je sposobnosti himera da se veže za oba receptora. Kao što je pronađeno za hCG vezanje za LH receptore i hSFH vezanje za FSH receptore, prisutnost čvora na ovim mjestima bila je puno više inhibitorna prema interakcijama FSH receptora nego prema interakcijama LH receptora. Ovo je pokazalo da su ovi ostaci bili puno bliži interface-u FSH receptora nego interface-u LH receptora. Prisustvo čvora na α-podjediničnim ostacima 48 i 52 je imalo puno manje utjecaja na interakcije himera s bilo kojim receptorom, ukazujući na to da su ovi ostaci dalje od receptorskog interface-a. Kao što je opservirano u primjerima 1 i 2, prisustvo čvora na α-podjediničnom ostatku 86 je bilo vrlo inhibitorno za vezanje himera za oba receptora. Ovo je pokazalo da je ova porcija hormona u blizini interface-a himera s oba receptora. Opservirani su slični rezultati sa čvorom na α-podjediničnom ostatku 91. As shown in Figure 12, the presence of a knot at α-subunit residue 35 did not interfere with the ability of the chimera to block the binding of radiodinated hCG or radiodinated FSH to the LH or FSH receptors, relative to each suitable bifunctional chimera lacking most of the carboxyterminal β- hCG subunits. However, the presence of a knot at α-subunit residue 37 led to a dramatically different result. Thus, this knot had very little effect on the chimera's ability to bind to LH receptors but almost eliminated its ability to bind to FSH receptors. The presence of a knot at α-subunit residues 43 and 46 reduced the ability of the chimera to bind to both receptors. As found for hCG binding to LH receptors and hSFH binding to FSH receptors, the presence of a node at these sites was much more inhibitory to FSH receptor interactions than to LH receptor interactions. This showed that these residues were much closer to the FSH receptor interface than to the LH receptor interface. The presence of a knot at α-subunit residues 48 and 52 had much less effect on the interactions of the chimeras with either receptor, indicating that these residues are further away from the receptor interface. As observed in Examples 1 and 2, the presence of a knot at α-subunit residue 86 was highly inhibitory to chimera binding to both receptors. This showed that this portion of the hormone is near the interface of the chimera with both receptors. Similar results were observed with the knot at α-subunit residue 91.
Kao što se može vidjeti na Slikama 13 i 14, prisustvo čvora reduciralo je aktivnost većine himera u LH receptorskim i FSH receptorskim signalnim transdukcijskim analizama. Ipak, količina redukcije se razlikovala zavisno od receptora s kojim je analog bio testiran. Na primjer, prisustvo čvora na α-podjediničnom ostatku 37 je bilo puno više inhibitorno na FSH receptorom izvabljenu akumulaciju cikličkog AMP-a, nego na LH receptorom posredovanu akumulaciju cikličkog AMP-a. Ukupno uzeto, relativni utjecaj čvorova na sposobnosti himera da izvabe celularni odgovor je sličan njihovim sposobnostima da utječu na interakcije receptora. Ove studije dovele su do zaključka da se interakcije lutropina kao što je hCG s LH receptorima lako razlikuju od onih od folitropina, kao što je hFSH s FSH receptorima. Dakle, iako lutropini I folitropini imaju vrlo slične strukture i iako su njihovi receptori vrlo slični, pronađeno je da način na koji ovi ligandi interreagiraju sa receptorima nije identičan. Ovaj primjer ilustrira moć upotrebljavanja testera predloženog izuma u identifikaciji protein-proteinskih interakcija. As can be seen in Figures 13 and 14, the presence of the node reduced the activity of most chimeras in the LH receptor and FSH receptor signal transduction assays. However, the amount of reduction differed depending on the receptor with which the analog was tested. For example, the presence of a knot at α-subunit residue 37 was much more inhibitory on FSH receptor-induced accumulation of cyclic AMP than on LH receptor-mediated accumulation of cyclic AMP. Taken together, the relative influence of nodes on the ability of chimeras to elicit a cellular response is similar to their ability to influence receptor interactions. These studies led to the conclusion that the interactions of lutropins such as hCG with LH receptors are readily distinguishable from those of follitropins such as hFSH with FSH receptors. Thus, although lutropins and follitropins have very similar structures and although their receptors are very similar, it was found that the way in which these ligands interact with the receptors is not identical. This example illustrates the power of using the tester of the present invention in the identification of protein-protein interactions.
Primjer 4 Example 4
Čvorovi koji imaju ß-laktamazu Nodules that have ß-lactamase
Analozi koji sadrže hCGß,S138C ß-podjedinice imaju relativno male čvorove koji se sastoje od ostataka što okružuju Ser138. Oni su dovoljno veliki da detektiraju udaljenosti između liganda i njegovog receptora koje su relativno male. Međutim, ovi čvorovi su premali da bi bili korisni za detekciju blizine ostataka koji su dalje od receptora. Ovo ograničenje se može zaobići povećavanjem veličine testera, što je učinjeno dodavanjem ß-laktamaze. ß-laktamaza je izabrana za upotrebu kao tester zbog njezine kristalne strukture koja je poznata i zato što se njezini aminoterminalni i karboksiterminalni krajevi nalaze na površini proteina. Prema tome, korisna je za generiranje fuzijskih proteina na bilo kojem kraju hormona. Još jedna prednost ß-laktamaze jest da je to enzim s visokim brojem prevrtanja koji cijepa fluorescentne supstrate (Zlokarnik, G., Neulescu, P. A., Knapp, T. E., Mere, L., Burres, N., Feng, L., Whitney, M., Roemer, K. & Tsien, R. Y. (1998) Science 279, 84-88), fenomen koji bi bio koristan za detekciju fuzijskih proteina. U ovom pogledu ß-laktamaza je upotrijebljena kao efikasan reporter (Moore, J. T., Davis, S. T. & Dev, I. K. (1997) Anal. Biochem. 247, 203-209), premda u vrlo različitom kontekstu nego što je ovdje zamišljeno. ß-laktamaza je također inhibirana proteinom koji se za nju veže (Strynadka, N. C., Jensen, S. E., Johns, K., Blanchard, H., Page, M., matagne, A., frere, J. M. & James, M. N. (1994) Nature 368, 657-660). Prema tome, bilo bi moguće povećati veličinu čvora ß-laktamaze povrh toga jednostavno dodavanjem inhibitora. Analogues containing hCGß,S138C ß-subunits have relatively small knots consisting of residues surrounding Ser138. They are large enough to detect distances between the ligand and its receptor that are relatively small. However, these nodes are too small to be useful for detecting the proximity of residues further away from the receptor. This limitation can be circumvented by increasing the size of the tester, which is done by adding ß-lactamase. ß-lactamase was chosen for use as a tester because of its crystal structure, which is known, and because its amino-terminal and carboxy-terminal ends are located on the surface of the protein. Therefore, it is useful for generating fusion proteins at either end of a hormone. Another advantage of ß-lactamase is that it is a high-turnover enzyme that cleaves fluorescent substrates (Zlokarnik, G., Neulescu, P. A., Knapp, T. E., Mere, L., Burres, N., Feng, L., Whitney, M., Roemer, K. & Tsien, R. Y. (1998) Science 279, 84-88), a phenomenon that would be useful for the detection of fusion proteins. In this respect ß-lactamase has been used as an efficient reporter (Moore, J.T., Davis, S.T. & Dev, I.K. (1997) Anal. Biochem. 247, 203-209), albeit in a very different context than intended here. ß-lactamase is also inhibited by its binding protein (Strynadka, N. C., Jensen, S. E., Johns, K., Blanchard, H., Page, M., matagne, A., frere, J. M. & James, M. N. (1994 ) Nature 368, 657-660). Therefore, it would be possible to increase the size of the ß-lactamase node on top of that simply by adding an inhibitor.
Pripremljena su dva derivata hCGß,S138C koja sadržavaju ß -laktamazu fuzijski protein. Jedan, poznat kao hCGß,S138C- ßLA (dugi) (SEQ ID NO: 44) ili jednostavnije DUGI, sadrži ß-laktamazu fuziranu za karboksiterminalni kraj hCGß,S138C kao što je prikazano na Slici 15. Ovaj protein je pripremljen PCR mutagenezom koristeći pUC18 kao kalup. Drugi tester poznat kao hCGß,S138C- ßLA (kratki) (SEQ ID NO:43) ili jednostavnije KRATKI, sadrži ß-laktamazu fuziranu za okrnjenu verziju hCGß,S138C kao što je prikazano na Slici 15. Protein KRATKI ima samo jednu aminokiselinu između sparivajućeg cisteina i početka ß-laktamaze. Očigledno, mogle bi se napraviti druge verzije ovih u kojima je različiti broj ostataka smješten između sparivajućeg cisteina i ß-laktamaze. Također bi trebalo prepoznati da bi onaj tko je iskusan u struci molekularnog modeliranja i molekularne biologije mogao dizajnirati analog ß-laktamaze u kojeg bi površinski ostatak bio zamijenjen cisteinom. Ovaj cistein može poslužiti kao sparivajući cistein, fenomen koji bi držao čvor ß-laktamaze u puno rigidnijoj vezi s ciljanim cisteinom nego što bi bilo moguće koristeći rep. Two derivatives of hCGß,S138C containing ß-lactamase fusion protein were prepared. One, known as hCGß,S138C- ßLA (long) (SEQ ID NO: 44) or simply LONG, contains a ß-lactamase fused to the carboxy-terminal end of hCGß,S138C as shown in Figure 15. This protein was prepared by PCR mutagenesis using pUC18 like a mold. Another probe, known as hCGß,S138C- ßLA (short) (SEQ ID NO:43) or simply SHORT, contains a ß-lactamase fused to a truncated version of hCGß,S138C as shown in Figure 15. The SHORT protein has only one amino acid between the mating cysteine and the beginning of ß-lactamase. Obviously, other versions of these could be made in which a different number of residues are placed between the pairing cysteine and the ß-lactamase. It should also be recognized that one skilled in the art of molecular modeling and molecular biology could design a ß-lactamase analog in which the surface residue is replaced by a cysteine. This cysteine can serve as a pairing cysteine, a phenomenon that would keep the ß-lactamase knot in a much more rigid bond with the target cysteine than would be possible using the tail.
Pet različitih acido stabilnih heterodimera je proizvedeno sadržavajući podjedinice DUGI i KRATKI. Oni su napravljeni da determiniraju relativnu blizinu α-podjediničnih ostataka 46, 48, 52, 64, i 92 prema LH receptoru. Kao što je prikazano na Slikama 16 i 17 niti jedan od ovih analoga nije bio aktivan kao hCG. Heterodimeri u kojih su DUGI i KRATKI β-podjedinični spleteni testeri spareni α-podjedinični ostatak 52 [i. e., αN52C + hCGß,S138C-ßLA (dugi) i αN52C + hCGß,S138C-ßLA (kratki), svaki ponaosob] je zadržao dosta aktivnosti hCG-a u signalnim transdukcijskim analizama. Mijenjanje α-podjediničnog ostatka Asn52 u cistein uzrokuje disrupciju glikozilacijskog signala i rezultira gubitkom oligosaharida na α-podjediničnoj petlji 2. Uklanjanje ovog oligosaharida sa hCG-a uzrokuje njegov gubitak efikasnosti od aproksimativno 60% (vidi Sliku 18). Prema tome, dodavanje čvora β-laktamaze na ovo mjesto može nadoknaditi gubitak efikasnosti uzrokovan uklanjanjem oligosaharida sa petlje 2. Relativno visoka aktivnost obaju ovih DUGOG i KRATKOG analoga pokazuje da α-podjedinični ostatak nije u blizini receptorskog interface-a. S obzirom da oba analoga imaju visoku efikasnost, ova otkrića impliciraju na ulogu oligosaharida u hCG-induciranoj signalnoj transdukciji. Aktivnosti DUGOG i KRATKOG analoga indiciraju da uloga oligosaharida na α-podjediničnoj petlji 2 jest da izvrnu pozicije unutar hCG-a; fenomen koji ne ovisi o specifičnim kontaktima između oligosaharida i bilo koje podjedinice. Five different acid-stable heterodimers were produced containing LONG and SHORT subunits. These were made to determine the relative proximity of α-subunit residues 46, 48, 52, 64, and 92 to the LH receptor. As shown in Figures 16 and 17, none of these analogs were as active as hCG. Heterodimers in which LONG and SHORT β-subunit braided testers are paired α-subunit residue 52 [i. e., αN52C + hCGß,S138C-ßLA (long) and αN52C + hCGß,S138C-ßLA (short), each individually] retained considerable hCG activity in signal transduction assays. Changing the α-subunit residue Asn52 to cysteine causes disruption of the glycosylation signal and results in the loss of the oligosaccharide on the α-subunit loop 2. Removal of this oligosaccharide from hCG causes its loss of efficiency of approximately 60% (see Figure 18). Therefore, the addition of a β-lactamase knot at this site can compensate for the loss of efficiency caused by the removal of the oligosaccharide from loop 2. The relatively high activity of both these LONG and SHORT analogs indicates that the α-subunit residue is not near the receptor interface. Given that both analogs have high efficacy, these findings implicate the role of oligosaccharides in hCG-induced signal transduction. The activities of the LONG and SHORT analogs indicate that the role of the oligosaccharides on α-subunit loop 2 is to reverse positions within hCG; a phenomenon that does not depend on specific contacts between the oligosaccharide and any subunit.
Aktivnosti preostalih analoga DUGOG i KRATKOG pokazuju da su α-podjedinični ostaci 48 i 64 puno bliže receptoru nego što bi se moglo determinirati preko aktivnosti hCGßS138C+αL48C i hCGßS138C+αS64C. Ovo otkriće ima važne konsekvence za modele hCG-LH receptorskog kompleksa. Ono također potvrđuje ranije nalaze da α-podjedinični ostaci jesu blizu receptorskom interface-u. The activities of the remaining LONG and SHORT analogs show that the α-subunit residues 48 and 64 are much closer to the receptor than could be determined by the activities of hCGßS138C+αL48C and hCGßS138C+αS64C. This finding has important consequences for models of the hCG-LH receptor complex. It also confirms earlier findings that α-subunit residues are close to the receptor interface.
Primjer 5 Example 5
Čvorovi koji imaju sparivajući cistein na aminoterminalnom kraju α-podjedinice Nodes that have a pairing cysteine at the amino-terminal end of the α-subunit
Nije neophodno da sparivajući cistein bude na karboksiterminalnom kraju proteina, te su dizajnirani su analozi u kojih je sparivajući cistein na aminoterminalnom kraju proteina. Aminokiselinska sekvenca jednog takvog analoga (ß101- 145,α) je ilustrirana kasnije u tekstu. Ovaj analog je pripremljen brisanjem ostataka 3-100 ß-podjedinice hCG-a i fuzijom α-podjedinice za kraj preostale ß-podjedinice koristeći metode standardnog PCR-a i kazetne mutageneze. Ima slobodni cistein na ostatku 12 koji služi kao sparivajući cistein. It is not necessary for the pairing cysteine to be at the carboxy-terminal end of the protein, and analogs have been designed in which the pairing cysteine is at the amino-terminal end of the protein. The amino acid sequence of one such analog (ß101-145,α) is illustrated later in the text. This analog was prepared by deleting residues 3-100 of the ß-subunit of hCG and fusing the α-subunit to the end of the remaining ß-subunit using standard PCR and cassette mutagenesis methods. It has a free cysteine at residue 12 that serves as a pairing cysteine.
[image] [image]
Primjer 6 Example 6
Metoda za produkciju mjesto-specifičnih proteinskih čvorova A method for the production of site-specific protein knots
Odabran je protein za kojeg se čvor treba pričvrstiti. Zatim, determinirana je specifična lokacija na proteinu za etiketiranje. Koristeći tehnike mutageneze poznate u struci, konstrukcija sposobna za ekspresiju proteina može biti pripremljena tako da je cisteinski ostatak supstituiran umjesto nativnog ostatka na specifičnoj lokaciji za etiketiranje u enkodiranog proteina. U dodatku, protein enkodiran u konstrukciji će se nadalje sastojati od porcije repa koja ima cisteinski ostatak pričvršćen za jedan kraj proteina, i čvora pričvršćenog za kraj porcije repa. Konstrukcija je tada umetnuta u stanicu zbog ekspresije rezultirajući produkcijom proteina u kojeg je čvor pričvršćen na specifičnom mjestu. The protein to which the node should be attached is selected. Next, the specific location on the protein for labeling was determined. Using mutagenesis techniques known in the art, a construct capable of protein expression can be prepared such that a cysteine residue is substituted for the native residue at a specific tagging location in the encoded protein. In addition, the protein encoded in the construct will further consist of a tail portion having a cysteine residue attached to one end of the protein, and a knot attached to the end of the tail portion. The construct is then inserted into the cell for expression resulting in the production of a protein into which the knot is attached at a specific site.
Dodavanje čvora proteinu uključuje uvođenje cisteina na površinu gdje čvor treba biti lociran i uvođenje cisteina na mjesto na aminoterminalnom ili karboksiterminalnom kraju proteina tako da cistein na kraju proteina može formirati disulfid s onim koji je dodan na površinu. Formacija disulfida između ovih dvaju cisteina uzrokovati će da se kraj proteina koji sadrži cistein stabilizira na površini stvarajući čvor na ovom mjestu (vidi Sliku 9). Adding a knot to a protein involves introducing a cysteine to the surface where the knot is to be located and introducing a cysteine to a site at the amino-terminal or carboxy-terminal end of the protein so that the cysteine at the end of the protein can form a disulfide with the one added to the surface. The formation of a disulfide between these two cysteines will cause the cysteine-containing end of the protein to stabilize on the surface by forming a knot at this site (see Figure 9).
Kako bi se čvor mogao stvoriti, kraj proteina koji sadrži cistein trebao bi biti dostatne duljine da dozvoli svom cisteinu da formira disulfid sa cisteinom na mjestu koje će sadržavati čvor. Ovo može zahtijevati dodavanje porcije repa kao što je karboksiterminalni kraj ß-podjedinice hCG-a kao što je bio slučaj pri dodavanju proteinskog čvora hFSH-u. Kompozicija porcije repa može naširoko varirati i ne treba biti limitirana onom od ß-podjedinice hCG-a. Glavni zahtjevi porcije repa su da mora biti dovoljno duga da dosegne cistein na površini proteina koji definira lokaciju čvora i da joj manjkaju ostaci što je sprečavaju da dođe na mjesto. Ovo bi uključivalo ostatke karakteristične za transmembranske domene i ostatke koji stvaraju mjesta što se vežu za druge proteine ili za dio proteina što je udaljen od cisteina na površini proteina na čvoru. In order for a knot to form, the cysteine-containing end of the protein should be of sufficient length to allow its cysteine to form a disulfide with the cysteine at the site that will contain the knot. This may require the addition of a tail portion such as the carboxy-terminal end of the ß-subunit of hCG as was the case for the addition of the protein knot to hFSH. The composition of the tail portion can vary widely and need not be limited to that of the ß-subunit of hCG. The main requirements of the tail portion are that it must be long enough to reach the cysteine on the surface of the protein that defines the location of the knot and that it lacks residues that prevent it from coming into place. This would include residues characteristic of transmembrane domains and residues that create binding sites for other proteins or for a portion of the protein remote from the cysteine on the protein surface at the node.
Ostaci na bilo kojoj strani cisteina koji je na aminoterminalnom ili karboksiterminalnom kraju proteina formiraju čvor (vidi Sliku 9). Generalno govoreći, što je veći broj ostataka na ovom mjestu, čvor je veći. Najmanji čvor bi se sastojao samo od cisteina (vidi Sliku 10A). Ovi bi bili stvoreni inkorporacijom cisteina na kraj aminoterminala ili karboksiterminalni kraj spone i dodavanjem mjesta za cijepanje odmah pokraj cisteina (vidi Sliku 10A). Mjesto cijepanja bi trebalo sadržavati aminokiselinsku sekvencu za proteazu što se ne nalazi unutar drugih dijelova proteina. Veličina čvora može biti povećana na najmanje tri načina. Prvo, protein može biti fuziran za kraj proteina upotrijebljenog za konstrukciju čvora (vidi Sliku 10B). Kao što je ranije u tekstu opisano, ß-laktamaza je fuzirana za kraj proteina. Međutim, ß-laktamaza nije jedini protein koji je prikladan za ovu svrhu. Izbor ß-laktamaze kao testera je olakšan zbog njezine kristalne strukture, koja je pokazala da je njen aminoterminalni kraj lociran na njenoj površini, što je povoljna pozicija za konstruiranje fuzijskog proteina. Drugo, cijepanje spone pomoću proteaze može biti upotrijebljeno za za reduciranje spone i njezinog doprinosa čvoru. I treće, udaljenost između sparivajućeg cisteina fuzijskog proteinskog testera može varirati povećavanjem ili reduciranjem broja aminokiselina. Kao što je viđeno na primjeru hCG-a, kraća je udaljenost uzrokovala veću redukciju hormonske aktivnosti zahvaljujući činjenici da je ograničila pokretljivost čvora. Ovo je zadržalo čvor bliže interface-u između hormona i LH receptora. Residues on either side of a cysteine that is at the amino-terminal or carboxy-terminal end of the protein form a knot (see Figure 9). Generally speaking, the greater the number of residues at this site, the larger the node. The smallest node would consist of only cysteines (see Figure 10A). These would be created by incorporating a cysteine at the amino-terminal or carboxy-terminal end of the linker and adding a cleavage site immediately adjacent to the cysteine (see Figure 10A). The cleavage site should contain an amino acid sequence for the protease that is not found within other parts of the protein. The size of the node can be increased in at least three ways. First, the protein can be fused to the end of the protein used to construct the knot (see Figure 10B). As described earlier in the text, ß-lactamase is fused to the end of the protein. However, ß-lactamase is not the only protein suitable for this purpose. The choice of ß-lactamase as a tester was facilitated by its crystal structure, which showed that its amino-terminal end is located on its surface, which is a favorable position for constructing a fusion protein. Second, cleavage of the bond by protease can be used to reduce the bond and its contribution to the knot. And third, the distance between the pairing cysteine of the fusion protein probe can be varied by increasing or decreasing the number of amino acids. As seen with the example of hCG, the shorter distance caused a greater reduction in hormonal activity due to the fact that it limited the mobility of the nodule. This kept the node closer to the interface between the hormone and the LH receptor.
Kao što bi trebalo biti evidentno iz Slike 10, nije esencijalno da sparivajući cistein bude prisutan u porciji repa. Uvođenje cisteina na površinu fuzijskog proteina koji je dio čvora gdje može formirati disulfid sa ciljanim cisteinom na proteinu trebalo bi uzrokovati direktno pričvršćivanje fuzijskog proteina na površinu proteina koji će biti testiran (vidi Sliku 10C). Ova metoda može isto tako biti upotrijebljena za kontroliranje orijentacije fuzijskog proteina koji je dio porcije čvora. Kada se proteaze upotrebljavaju kao fuzijski proteini, uvođenje cisteina na površinu daleko od aktivnog mjesta držati će aktivno mjesto fuzijskog proteina od površine za koju se pričvršćuje čvor. As should be evident from Figure 10, it is not essential that the pairing cysteine be present in the tail portion. Introducing a cysteine to the surface of the fusion protein that is part of the node where it can form a disulfide with the target cysteine on the protein should cause direct attachment of the fusion protein to the surface of the protein to be tested (see Figure 10C). This method can also be used to control the orientation of a fusion protein that is part of the node portion. When proteases are used as fusion proteins, introducing cysteines on a surface away from the active site will keep the active site of the fusion protein away from the surface to which the knot attaches.
Primjer 7 Example 7
Upotreba skraćene porcije repa kao testera Using a shortened portion of the tail as a tester
Dodavanje čvorova na proteine dozvoljava procjenu udaljenosti između dviju proteinskih površina. Kao što je pokazano, znatne porcije α-podjedinične petlje 2 hCG-a ne kontaktiraju receptor. Lokacija sparivajućeg cisteina na amino kiselini 138 ß-podjedinice hCG-a postavlja relativno malo ograničenja na pozicije koje mogu biti zauzete sa ß-podjediničnim ostacima 111-137 koji vežu sparivajući cistein na čvoru sa jezgrom ß-podjedinice hCG-a. I sama porcija repa može biti upotrijebljena da testira proteinske površine, a postoji nekoliko slučajeva u kojima bi bilo poželjno ograničiti njezinu poziciju na posebne porcije molekule. Ove uključuju one u kojima bi bilo poželjno prekriti porciju aktivnog mjesta proteina tako ga čineći inaktivnim sve dok porcija repa nije izrezana s proteazom. Adding nodes to proteins allows estimation of the distance between two protein surfaces. As shown, substantial portions of the α-subunit loop 2 of hCG do not contact the receptor. The location of the pairing cysteine at amino acid 138 of the hCG ß-subunit places relatively little restriction on the positions that can be occupied by the ß-subunit residues 111-137 that bind the pairing cysteine at the junction with the hCG ß-subunit core. The tail portion itself can be used to probe protein surfaces, and there are several cases where it would be desirable to restrict its position to specific portions of the molecule. These include those in which it would be desirable to cover the active site portion of the protein thus rendering it inactive until the tail portion is excised with a protease.
U studijama o hCG-LH receptorskim interakcijama, bilo je poželjno ograničiti lokaciju porcije repa na brazdu između α-podjedinične petlje 2 i ß-podjediničnih petlji 1 i 3, kako bi se dokazala tvrdnja da je ova brazda formirala ključni receptorski kontakt. Ranije opisana upotreba porcije repa bi joj dozvolila da prođe kroz ovu porciju hormona ali je ne bi prisiljavala da tamo bude locirana. Zahvaljujući svojoj duljini, porcija repa je možda prešla preko konveksne površine ß-podjediničnih petlji 1 i 3 kako bi omogućila sparivajućem cisteinu čvora da dosegne brojne cisteine koji su supstituirani umjesto ostataka u α-podjediničnoj petlji 2. Iz tog razloga, skratili smo porciju repa kako bi je prisilili da prođe kroz brazdu između α-podjedinične petlje 2 i ß-podjediničnih petlji 1 i 3 kada ciljamo na cistein koji je supstituiran umjesto ostataka 42, 46, ili 48 α-podjedinice. Analozi porcije repa hCGß,δ116-135,S138C (SEQ ID NO: 45) i hCGß,δ121-135,S138C (SEQ ID NO: 46) (vidi Sliku 15) su bili prekratki da omoguće formiranje disulfida između čvora i ciljanog proteina osim ako anlozi porcije repa ne prođu kroz brazdu između α-podjedinične petlje 2 i ß-podjediničnih petlji 1 i 3. Kao što se može vidjeti iz rezultata prikazanih na Slikama 23 i 24, heterodimeri što sadrže αT46C i hCGß,δ116-135,S138C ili hCGß,δ121-135,S138C su imali znatnu receptor vežuću aktivnost. I heterodimer koji sadržava αL48C i hCGß,δ121-135,S138C je također imao značajnu receptor vežuću aktivnost. Formirane su puno manje količine acido stabilnog heterodimera koji sadržava αL48C i hCGß,δ116-135,S138C, upućujući na to da najkraća porcija repa koja je ispitivana možda nije bila dovoljne duljine da sparivajući cistein čvora dosegne ciljani cistein na proteinu. S obzirom na to da porciji repa ovih analoga nije bilo neophodno da prođe kroz brazdu između α-podjedinične petlje 2 i ß-podjediničnih petlji 1 i 3 kako bi sparivajući cistein čvora dosegao ciljani cistein na α-podjediničnom ostatku 42, ovaj analog je upotrijebljen kao pozitivna kontrola. In studies of hCG-LH receptor interactions, it was desirable to limit the location of the tail portion to the groove between the α-subunit loop 2 and the ß-subunit loops 1 and 3, to substantiate the claim that this groove formed a key receptor contact. The use of the portion of the tail described earlier would allow her to pass through this portion of hormones but would not force her to be located there. Due to its length, the tail portion may have crossed over the convex surface of the ß-subunit loops 1 and 3 to allow the pairing cysteine of the knot to reach the numerous cysteines substituted for residues in the α-subunit loop 2. For this reason, we shortened the tail portion as would force it to pass through the groove between α-subunit loop 2 and ß-subunit loops 1 and 3 when targeting a cysteine substituted instead of residues 42, 46, or 48 of the α-subunit. The tail portion analogs of hCGß,δ116-135,S138C (SEQ ID NO: 45) and hCGß,δ121-135,S138C (SEQ ID NO: 46) (see Figure 15) were too short to allow disulfide formation between the node and the target protein except if the tail portion analogs do not pass through the groove between the α-subunit loop 2 and the ß-subunit loops 1 and 3. As can be seen from the results shown in Figures 23 and 24, heterodimers containing αT46C and hCGß,δ116-135,S138C or hCGß,δ121-135,S138C had significant receptor binding activity. And the heterodimer containing αL48C and hCGß,δ121-135,S138C also had significant receptor binding activity. Much smaller amounts of an acid-stable heterodimer containing αL48C and hCGß,δ116-135,S138C were formed, suggesting that the shortest portion of the tail examined may not have been of sufficient length for the pairing cysteine of the knot to reach the target cysteine on the protein. Given that the tail portion of these analogs did not need to pass through the groove between α-subunit loop 2 and ß-subunit loops 1 and 3 in order for the pairing cysteine of the node to reach the target cysteine at α-subunit residue 42, this analog was used as positive control.
Sveukupno uzevši, ove opservacije su pokazale da brazda nije sudjelovala u esencijalnim receptorskim kontaktima. Također je demonstrirano kako pozicija porcije repa može biti manipulirana i tako joj se omogućilo da prođe blizu specifičnih porcija na proteinu. Ovo bi dozvolilo da se porcija repa uptrijebi za prikrivanje specifičnog mjesta, svojstvo koje kad se kombinira sa mjestima cijepanja koja su uvedena u porciju repa može biti korisno za pripremanje latentnih proteaza, toksina, ili drugih korisnih analoga. Porcija repa koja skriva mjesto proteaze ili toksina bi dozvolila pripremanje i upotrebu reagensa sposobnih za ulaženje u stanice gdje bi endogene ili druge proteaze cijepale porciju repa da aktiviraju toksin. Ovi agensi bi bili korisni kao terapeutici za liječenje malignoma ili drugih bolesti. Taken together, these observations indicated that the furrow was not involved in essential receptor contacts. It was also demonstrated that the position of the tail portion can be manipulated to allow it to pass close to specific portions on the protein. This would allow the tail portion to be used to mask a specific site, a property that when combined with cleavage sites introduced into the tail portion may be useful for preparing latent proteases, toxins, or other useful analogs. A tail portion that conceals a protease or toxin site would permit the preparation and use of reagents capable of entering cells where endogenous or other proteases would cleave the tail portion to activate the toxin. These agents would be useful as therapeutics for the treatment of malignancies or other diseases.
Primjer 8 Example 8
Upotreba testera pričvršćenih za ß-podjedinicu Use of probes attached to the ß-subunit
Sugerirano je da mala petlja na sigurnosnom pojasu ima ulogu u biološkoj aktivnosti hCG-a, te je bilo poželjno pričvrstiti čvorove na ovu regiju ß-podjedinice kako bi se istražila ova mogućnost. Producirani su analozi alfa podjedinice koji su sadržavali karboksiterminalne ostatke u ß-podjedinici hCG-a. Već je prikazano kako je pričvršćavanje porcije karboksiterminala ß-podjedinice hCG-a za karboksiterminalni kraj α-podjedinice reduciralo aktivnost heterodimera za 50-pregiba ili više, međutim, upotreba cijele ß-podjedinične sekvence nije imala takvu posljedicu. Analozi koji sadrže cjelokupne karboksiterminalne ostatke ß-podjedinice hCG-a pričvršćene za α-podjedinicu, uključujući aminokiseline Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln, imali su 50% ili više aktivnosti hCG-a u receptor vežućim i signalnim analizama. Otkriveno je da se to može zahvaliti prisutnosti nabijenih ostataka u blizini dodirne točke ostataka deriviranih iz α-podjedinice i ß-podjedinice. Kako bi se pričvrstio čvor za ß-podjedinične ostatke na maloj petlji sigurnosnog pojasa, okrnjena verzija karboksiterminala ß-podjedinice hCG-a je dodana na kraj α-podjedinice za stvaranje hCG-αCTδ116/135,138C (SEQ ID NO: 57). On je ko-eksprimiran u COS-7 stanicama sa hCG ß-podjediničnim analogom što ima cistein na mjestu Arg94 (SEQ ID NO: 58), Arg95 (SEQ ID NO: 59), Ser96 (SEQ ID NO: 60), Thr97 (SEQ ID NO: 61), Thr98 (SEQ ID NO: 62), i Asp99 (SEQ ID NO: 63). Svi su ovi proteini formirali acido-stabilne heterodimere i kao što se može vidjeti iz podataka prikazanih na Slikama 26, 27, 28, 29, 30, 31, i 32, ove su studije otkrile kako ovaj dio ß-podjedinice interreagira sa LH receptorima (LHR). Prisutnost ovog čvora na ostacima 95 i 99 eliminirala je LHR interakciju i biološku aktivnost. Ovaj čvor imao je puno manji utjecaj kad je bio prisutan na ostacima 96 ili 97 jer su ovi analozi imali znatnu aktivnost u vežućim i signalnim analizama. Ovaj čvor je reducirao receptorske interakcije više kada je bio pričvršćen za ostatak 98, ali ne ni blizu kao što je viđeno kad je bio pričvršćen za ostatke 95 ili 99. Ovo sugerira da su potonji analozi možda locirani bliže receptorskom interface-u. A small loop in the seat belt has been suggested to play a role in the biological activity of hCG, and it was desirable to attach knots to this region of the ß-subunit to investigate this possibility. Analogues of the alpha subunit were produced that contained the carboxy-terminal residues in the ß-subunit of hCG. It has already been shown that attachment of the carboxy-terminal portion of the ß-subunit of hCG to the carboxy-terminal end of the α-subunit reduced heterodimer activity by 50-fold or more, however, use of the entire ß-subunit sequence had no such effect. Analogues containing the entire carboxy-terminal residues of the ß-subunit of hCG attached to the α-subunit, including the amino acids Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro- Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln, had 50% or more hCG activity in receptor binding and signaling analyses. It was found that this could be due to the presence of charged residues near the point of contact of residues derived from the α-subunit and the ß-subunit. To attach the knot to the ß-subunit residues on the small loop of the seat belt, a truncated version of the carboxy-terminal ß-subunit of hCG was added to the end of the α-subunit to create hCG-αCTδ116/135,138C (SEQ ID NO: 57). It was co-expressed in COS-7 cells with an hCG ß-subunit analog having a cysteine at Arg94 (SEQ ID NO: 58), Arg95 (SEQ ID NO: 59), Ser96 (SEQ ID NO: 60), Thr97 ( SEQ ID NO: 61), Thr98 (SEQ ID NO: 62), and Asp99 (SEQ ID NO: 63). All of these proteins formed acid-stable heterodimers and as can be seen from the data shown in Figures 26, 27, 28, 29, 30, 31, and 32, these studies revealed how this part of the ß-subunit interacts with LH receptors ( LHR). The presence of this knot at residues 95 and 99 eliminated LHR interaction and biological activity. This node had much less impact when present at residues 96 or 97 because these analogs had significant activity in binding and signaling assays. This knot reduced receptor interactions more when attached to residue 98, but not nearly as much as seen when attached to residues 95 or 99. This suggests that the latter analogs may be located closer to the receptor interface.
Postranični lanac hCG ß-podjediničnog ostatka 95 okrenut je u suprotnom smjeru od onih od ostataka 94 i 96, fenomen koji je možda odgovoran za činjenicu da je pričvršćivanje čvora na ovo mjesto djelovalo puno više inhibitorno na hCG-LHR interakcije nego nego pričvršćivanje čvora na ostatke 94 ili 96. Ovo sugerira da je površina sigurnosnog pojasa što je u blizini postraničnog lanca od Arg95 možda blizu receptorskog interface-a. Kako bi saznali da li postranični lanac kontaktira s površinom hormona, pripremljeni su analozi koji su sadržavali manje čvorove. Ovi su bili konstruirani zamjenom serina na α-podjediničnom ostatku 92 sa cisteinom (SEQ ID NO: 35) ili dodavanjem repa sastavljenog od Gly-Gly-Cys na karboksiterminal α-podjedinice (SEQ ID NO: 64). Kao što se može vidjeti na Slikama 30 i 32, ovi manji čvorovi su imali manju sposobnost da interferiraju sa aktivnošću heterodimera u LHR analizama. Prema tome, oba acido-stabilna ukrižena heterodimera su zadržala poprilične aktivnosti u ovim analizama. Ovim je pokazano da je vjerojatno da je postranični lanac hCG ß-podjediničnog ostatka 95 u blizini receptorskog interface-a, ali da nije potreban za esencijalne receptorske kontakte. Ovim je isto tako pokazano da je lokacija karboksiterminalnog kraja α-podjedinice u blizini male petlje sigurnosnog pojasa nakon što hormon interreagira sa LHR. The side chain of the hCG ß-subunit residue 95 faces in the opposite direction to those of residues 94 and 96, a phenomenon that may be responsible for the fact that knot attachment at this site was much more inhibitory to hCG-LHR interactions than knot attachment at residues 94 or 96. This suggests that the seat belt area adjacent to the side chain of Arg95 may be close to the receptor interface. In order to find out whether the side chain contacts the surface of the hormone, analogues containing smaller knots were prepared. These were constructed by replacing serine at α-subunit residue 92 with cysteine (SEQ ID NO: 35) or by adding a tail composed of Gly-Gly-Cys to the carboxy-terminus of the α-subunit (SEQ ID NO: 64). As can be seen in Figures 30 and 32, these smaller nodes had less ability to interfere with heterodimer activity in LHR assays. Therefore, both acid-stable cross-linked heterodimers retained considerable activity in these assays. This demonstrated that the side chain of the hCG ß-subunit residue 95 is likely to be near the receptor interface, but not required for essential receptor contacts. This also shows that the location of the carboxy-terminal end of the α-subunit is near the small loop of the seat belt after the hormone interacts with LHR.
S obzirom da je α-podjedinični karboksiterminal potreban za punu aktivnost glikoproteinskog hormona, kao što je oligosaharid α-podjedinične petlje 2 na Asn52, bilo je od velikog interesa vidjeti i determinirati kako je mijenjanje pozicije α-podjediničnog karboksiterminala u heterodimera utjecalo na njegovu biološku aktivnost. Pripremljen je analog α-podjedinice hCG-a koji ne može biti glikoziliran na α-podjediničnoj petlji 2 zahvaljujući promjeni Asn52 u Asp i činjenici da ima sposobnost dodavanja cisteinskog čvora na β-podjedinične ostatke 92, 94, 95, i 96 (SEQ ID NO: 65). Ko-ekspresija ovog analoga u COS-7 stanicama sa β-podjediničnim analozima što sadrže cistein na ostacima 92, 94, 95, i 96 , dovela je do formacije acido-stabilnih ukriženih heterodimera. Aktivnosti ovih analoga i himera β-podjediničnog analoga koji je sadržavao cistein na mjestu Arg96 (SEQ ID NO: 66) su pokazale da karboksiterminal α-podjedinice može biti pričvršćen na nekoliko mjesta u β-podjedinici, ukazujući na to da njegova lokacija nije neprikladno ograničena (vidi Sliku 33). Nadalje, nalaz da je efikasnost analoga himera bila niska pokazao je da konformacija ove porcije hormona ima utjecaj na signalnu transdukciju. Given that the α-subunit carboxyterminal is required for full glycoprotein hormone activity, such as the α-subunit loop 2 oligosaccharide at Asn52, it was of great interest to see and determine how changing the position of the α-subunit carboxyterminal in the heterodimer affected its biological activity . An analog of the α-subunit of hCG was prepared that cannot be glycosylated on the α-subunit loop 2 thanks to the change of Asn52 to Asp and the fact that it has the ability to add a cysteine knot to the β-subunit residues 92, 94, 95, and 96 (SEQ ID NO : 65). Co-expression of this analog in COS-7 cells with β-subunit analogs containing cysteine at residues 92, 94, 95, and 96 led to the formation of acid-stable cross-linked heterodimers. The activities of these analogs and a chimeric β-subunit analog containing a cysteine at Arg96 (SEQ ID NO: 66) indicated that the carboxy terminus of the α-subunit can be attached to several sites in the β-subunit, indicating that its location is not unduly constrained. (see Figure 33). Furthermore, the finding that the efficiency of the chimera analogue was low showed that the conformation of this portion of the hormone has an influence on signal transduction.
Netko iskusan u struci brzo će procijeniti predloženi izum, da je dobro adaptiran da provede svrhu i poluči konačni cilj, te spomenute prednosti kao i one koje su prema tome inherentne. Kompozicije zajedno s metodama i procedurama ovdje opisanim su trenutačno reprezentativne za pretpostavljena ostvarenja i primjerena su, te nisu namijenjena za ograničavanje polja djelovanja ovog izuma. Prema tome, iskusnima u struci koji su upućeni u sami smisao izuma će se javiti ideje o promjenama i drugim načinima upotrebe, definiranim njegovim poljem djelovanja i zahtjevima. One skilled in the art will readily appreciate the proposed invention as being well adapted to carry out the purpose and achieve the ultimate goal, and the aforementioned advantages as well as those inherent thereto. The compositions together with the methods and procedures described herein are presently representative of the contemplated embodiments and are appropriate, and are not intended to limit the scope of the present invention. Therefore, those skilled in the art who are familiar with the very meaning of the invention will have ideas about changes and other ways of use, defined by its field of operation and requirements.
Iskusnima u struci bit će isto tako uskoro očigledno da različite supstitucije i modifikacije mogu biti učinjene na ovdje obznanjenom izumu, a bez napuštanja polja djelovanja i smisla samog izuma. It will also soon be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein, without departing from the scope and spirit of the invention itself.
Svi patenti i publikacije koji su ovdje referencirani su inkorporirani referencom u istom opsegu kao da je za svaku individualnu publikaciju specifično i individualno indicirano da bude inkorporirana referencom. Popis referenci koji slijedi je također inkorporiran referencom. All patents and publications referenced herein are incorporated by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. The following list of references is also incorporated by reference.
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| CN112986571B (en) * | 2019-12-12 | 2023-04-11 | 中国科学院大连化学物理研究所 | Method for identifying Midkine spatiotemporal network interaction protein |
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| IL161804A0 (en) | 2005-11-20 |
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