CN107531770A - Met受体激动蛋白 - Google Patents
Met受体激动蛋白 Download PDFInfo
- Publication number
- CN107531770A CN107531770A CN201680006622.2A CN201680006622A CN107531770A CN 107531770 A CN107531770 A CN 107531770A CN 201680006622 A CN201680006622 A CN 201680006622A CN 107531770 A CN107531770 A CN 107531770A
- Authority
- CN
- China
- Prior art keywords
- protein
- ser
- gly
- met
- glu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000008484 agonism Effects 0.000 title description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 155
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 154
- 230000004913 activation Effects 0.000 claims abstract description 23
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 claims abstract description 17
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims description 94
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 67
- 150000001413 amino acids Chemical group 0.000 claims description 23
- 150000007523 nucleic acids Chemical class 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 19
- 230000001684 chronic effect Effects 0.000 claims description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 16
- 201000010099 disease Diseases 0.000 claims description 15
- 239000013604 expression vector Substances 0.000 claims description 14
- 108020004707 nucleic acids Proteins 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 13
- 208000024891 symptom Diseases 0.000 claims description 13
- 230000001154 acute effect Effects 0.000 claims description 12
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 9
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 206010072170 Skin wound Diseases 0.000 claims description 7
- 206010012601 diabetes mellitus Diseases 0.000 claims description 7
- 230000001537 neural effect Effects 0.000 claims description 7
- 210000000056 organ Anatomy 0.000 claims description 7
- 230000004083 survival effect Effects 0.000 claims description 7
- 208000030090 Acute Disease Diseases 0.000 claims description 6
- 101710198693 Invasin Proteins 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 238000002059 diagnostic imaging Methods 0.000 claims description 6
- 238000002405 diagnostic procedure Methods 0.000 claims description 6
- 210000003734 kidney Anatomy 0.000 claims description 6
- 208000019423 liver disease Diseases 0.000 claims description 6
- 208000031225 myocardial ischemia Diseases 0.000 claims description 6
- 208000033808 peripheral neuropathy Diseases 0.000 claims description 6
- 230000008929 regeneration Effects 0.000 claims description 6
- 238000011069 regeneration method Methods 0.000 claims description 6
- 208000019693 Lung disease Diseases 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 230000000451 tissue damage Effects 0.000 claims description 4
- 231100000827 tissue damage Toxicity 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
- 208000030613 peripheral artery disease Diseases 0.000 claims description 3
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 claims description 2
- 208000012902 Nervous system disease Diseases 0.000 claims 1
- 230000001737 promoting effect Effects 0.000 claims 1
- 210000005253 yeast cell Anatomy 0.000 claims 1
- 230000008520 organization Effects 0.000 abstract description 4
- 235000018102 proteins Nutrition 0.000 description 130
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 29
- 125000003275 alpha amino acid group Chemical group 0.000 description 27
- 230000026731 phosphorylation Effects 0.000 description 25
- 238000006366 phosphorylation reaction Methods 0.000 description 25
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 22
- 210000004185 liver Anatomy 0.000 description 21
- 235000001014 amino acid Nutrition 0.000 description 20
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 18
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 16
- 108010006664 gamma-glutamyl-glycyl-glycine Proteins 0.000 description 16
- 239000000370 acceptor Substances 0.000 description 15
- 108010015792 glycyllysine Proteins 0.000 description 15
- 239000012190 activator Substances 0.000 description 14
- 108010077245 asparaginyl-proline Proteins 0.000 description 14
- 230000000694 effects Effects 0.000 description 14
- PJOPLXOCKACMLK-KKUMJFAQSA-N Arg-Tyr-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O PJOPLXOCKACMLK-KKUMJFAQSA-N 0.000 description 13
- PRVVCRZLTJNPCS-FXQIFTODSA-N Cys-Arg-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CS)N)CN=C(N)N PRVVCRZLTJNPCS-FXQIFTODSA-N 0.000 description 13
- BWVHQINTNLVWGZ-ZKWXMUAHSA-N Val-Cys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)O)N BWVHQINTNLVWGZ-ZKWXMUAHSA-N 0.000 description 13
- 210000003000 inclusion body Anatomy 0.000 description 13
- 239000000178 monomer Substances 0.000 description 13
- 238000001262 western blot Methods 0.000 description 13
- YUOXLJYVSZYPBJ-CIUDSAMLSA-N Asn-Pro-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O YUOXLJYVSZYPBJ-CIUDSAMLSA-N 0.000 description 12
- BATWGBRIZANGPN-ZPFDUUQYSA-N Ile-Pro-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)N)C(=O)O)N BATWGBRIZANGPN-ZPFDUUQYSA-N 0.000 description 12
- XLILXFRAKOYEJX-GUBZILKMSA-N Asp-Leu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O XLILXFRAKOYEJX-GUBZILKMSA-N 0.000 description 11
- ZJICFHQSPWFBKP-AVGNSLFASA-N Glu-Asn-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZJICFHQSPWFBKP-AVGNSLFASA-N 0.000 description 11
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 11
- GNRMAQSIROFNMI-IXOXFDKPSA-N Phe-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GNRMAQSIROFNMI-IXOXFDKPSA-N 0.000 description 11
- JRBWMRUPXWPEID-JYJNAYRXSA-N Pro-Trp-Cys Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CS)C(=O)O)C(=O)[C@@H]1CCCN1 JRBWMRUPXWPEID-JYJNAYRXSA-N 0.000 description 11
- 108010057821 leucylproline Proteins 0.000 description 11
- 238000012545 processing Methods 0.000 description 11
- XMENRVZYPBKBIL-AVGNSLFASA-N His-Glu-His Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O XMENRVZYPBKBIL-AVGNSLFASA-N 0.000 description 10
- YNMQUIVKEFRCPH-QSFUFRPTSA-N Ile-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)O)N YNMQUIVKEFRCPH-QSFUFRPTSA-N 0.000 description 10
- QGXQHJQPAPMACW-PPCPHDFISA-N Ile-Thr-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)O)N QGXQHJQPAPMACW-PPCPHDFISA-N 0.000 description 10
- MLLKLNYPZRDIQG-GUBZILKMSA-N Lys-Cys-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N MLLKLNYPZRDIQG-GUBZILKMSA-N 0.000 description 10
- HZLSUXCMSIBCRV-RVMXOQNASA-N Met-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCSC)N HZLSUXCMSIBCRV-RVMXOQNASA-N 0.000 description 10
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 10
- MKGIILKDUGDRRO-FXQIFTODSA-N Pro-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 MKGIILKDUGDRRO-FXQIFTODSA-N 0.000 description 10
- IOVHBRCQOGWAQH-ZKWXMUAHSA-N Ser-Gly-Ile Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOVHBRCQOGWAQH-ZKWXMUAHSA-N 0.000 description 10
- UPLYXVPQLJVWMM-KKUMJFAQSA-N Ser-Phe-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UPLYXVPQLJVWMM-KKUMJFAQSA-N 0.000 description 10
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 239000000499 gel Substances 0.000 description 10
- 108010018006 histidylserine Proteins 0.000 description 10
- LKHMGNHQULEPFY-ACZMJKKPSA-N Cys-Ser-Glu Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O LKHMGNHQULEPFY-ACZMJKKPSA-N 0.000 description 9
- 229920002971 Heparan sulfate Polymers 0.000 description 9
- 238000009792 diffusion process Methods 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- PHHRSPBBQUFULD-UWVGGRQHSA-N Arg-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N PHHRSPBBQUFULD-UWVGGRQHSA-N 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 8
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 8
- 239000000556 agonist Substances 0.000 description 8
- 239000013078 crystal Substances 0.000 description 8
- 238000000605 extraction Methods 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 8
- 229920000669 heparin Polymers 0.000 description 8
- 229960002897 heparin Drugs 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 7
- OAGVHWYIBZMWLA-YFKPBYRVSA-N Glu-Gly-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)NCC(O)=O OAGVHWYIBZMWLA-YFKPBYRVSA-N 0.000 description 7
- AKXBNSZMYAOGLS-STQMWFEESA-N Tyr-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AKXBNSZMYAOGLS-STQMWFEESA-N 0.000 description 7
- 239000011324 bead Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- 241000282414 Homo sapiens Species 0.000 description 6
- VPBQDHMASPJHGY-JYJNAYRXSA-N Pro-Trp-Ser Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CO)C(=O)O VPBQDHMASPJHGY-JYJNAYRXSA-N 0.000 description 6
- HKHCTNFKZXAMIF-KKUMJFAQSA-N Ser-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CC1=CC=C(O)C=C1 HKHCTNFKZXAMIF-KKUMJFAQSA-N 0.000 description 6
- 239000012505 Superdex™ Substances 0.000 description 6
- ARKBYVBCEOWRNR-UBHSHLNASA-N Trp-Ser-Ser Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O ARKBYVBCEOWRNR-UBHSHLNASA-N 0.000 description 6
- KLGFILUOTCBNLJ-IHRRRGAJSA-N Tyr-Cys-Arg Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O KLGFILUOTCBNLJ-IHRRRGAJSA-N 0.000 description 6
- VTCKHZJKWQENKX-KBPBESRZSA-N Tyr-Lys-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O VTCKHZJKWQENKX-KBPBESRZSA-N 0.000 description 6
- 210000004899 c-terminal region Anatomy 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 239000013592 cell lysate Substances 0.000 description 6
- 239000000539 dimer Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 108010089804 glycyl-threonine Proteins 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 108010082117 matrigel Proteins 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000000700 radioactive tracer Substances 0.000 description 6
- 108010071207 serylmethionine Proteins 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 230000009182 swimming Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 108010051110 tyrosyl-lysine Proteins 0.000 description 6
- RWWPBOUMKFBHAL-FXQIFTODSA-N Arg-Asn-Cys Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(O)=O RWWPBOUMKFBHAL-FXQIFTODSA-N 0.000 description 5
- BECXEHHOZNFFFX-IHRRRGAJSA-N Arg-Ser-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BECXEHHOZNFFFX-IHRRRGAJSA-N 0.000 description 5
- CUXIOFHFFXNUGG-HTFCKZLJSA-N Cys-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](CS)N CUXIOFHFFXNUGG-HTFCKZLJSA-N 0.000 description 5
- FGWRYRAVBVOHIB-XIRDDKMYSA-N Gln-Pro-Trp Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)N)N)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O FGWRYRAVBVOHIB-XIRDDKMYSA-N 0.000 description 5
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 5
- RFMDODRWJZHZCR-BJDJZHNGSA-N Ile-Lys-Cys Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(O)=O RFMDODRWJZHZCR-BJDJZHNGSA-N 0.000 description 5
- BJECXJHLUJXPJQ-PYJNHQTQSA-N Ile-Pro-His Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N BJECXJHLUJXPJQ-PYJNHQTQSA-N 0.000 description 5
- 235000014852 L-arginine Nutrition 0.000 description 5
- CANPXOLVTMKURR-WEDXCCLWSA-N Lys-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN CANPXOLVTMKURR-WEDXCCLWSA-N 0.000 description 5
- SBQDRNOLGSYHQA-YUMQZZPRSA-N Lys-Ser-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SBQDRNOLGSYHQA-YUMQZZPRSA-N 0.000 description 5
- QGQGAIBGTUJRBR-NAKRPEOUSA-N Met-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCSC QGQGAIBGTUJRBR-NAKRPEOUSA-N 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 5
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 5
- AABIBDJHSKIMJK-FXQIFTODSA-N Ser-Ser-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O AABIBDJHSKIMJK-FXQIFTODSA-N 0.000 description 5
- AHHJARQXFFGOKF-NRPADANISA-N Val-Glu-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N AHHJARQXFFGOKF-NRPADANISA-N 0.000 description 5
- PGQUDQYHWICSAB-NAKRPEOUSA-N Val-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N PGQUDQYHWICSAB-NAKRPEOUSA-N 0.000 description 5
- 238000001042 affinity chromatography Methods 0.000 description 5
- 238000003016 alphascreen Methods 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 230000005540 biological transmission Effects 0.000 description 5
- 208000015114 central nervous system disease Diseases 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 239000003102 growth factor Substances 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- YKJYKKNCCRKFSL-RDBSUJKOSA-N (-)-anisomycin Chemical compound C1=CC(OC)=CC=C1C[C@@H]1[C@H](OC(C)=O)[C@@H](O)CN1 YKJYKKNCCRKFSL-RDBSUJKOSA-N 0.000 description 4
- YKJYKKNCCRKFSL-UHFFFAOYSA-N Anisomycin Natural products C1=CC(OC)=CC=C1CC1C(OC(C)=O)C(O)CN1 YKJYKKNCCRKFSL-UHFFFAOYSA-N 0.000 description 4
- QXOPPIDJKPEKCW-GUBZILKMSA-N Asn-Pro-Arg Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O QXOPPIDJKPEKCW-GUBZILKMSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000005636 Cyclic AMP Response Element-Binding Protein Human genes 0.000 description 4
- 108010045171 Cyclic AMP Response Element-Binding Protein Proteins 0.000 description 4
- QQOWCDCBFFBRQH-IXOXFDKPSA-N Cys-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CS)N)O QQOWCDCBFFBRQH-IXOXFDKPSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 4
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 4
- MLILEEIVMRUYBX-NHCYSSNCSA-N Glu-Val-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O MLILEEIVMRUYBX-NHCYSSNCSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- DKKGAAJTDKHWOD-BIIVOSGPSA-N Ser-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)C(=O)O DKKGAAJTDKHWOD-BIIVOSGPSA-N 0.000 description 4
- FGBLCMLXHRPVOF-IHRRRGAJSA-N Ser-Tyr-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FGBLCMLXHRPVOF-IHRRRGAJSA-N 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 210000002744 extracellular matrix Anatomy 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000001476 gene delivery Methods 0.000 description 4
- 210000003494 hepatocyte Anatomy 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000001542 size-exclusion chromatography Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- 230000029663 wound healing Effects 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 206010001497 Agitation Diseases 0.000 description 3
- AUFHLLPVPSMEOG-YUMQZZPRSA-N Arg-Gly-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AUFHLLPVPSMEOG-YUMQZZPRSA-N 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- WDQXKVCQXRNOSI-GHCJXIJMSA-N Cys-Asp-Ile Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WDQXKVCQXRNOSI-GHCJXIJMSA-N 0.000 description 3
- LWDGZZGWDMHBOF-FXQIFTODSA-N Gln-Glu-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O LWDGZZGWDMHBOF-FXQIFTODSA-N 0.000 description 3
- SOEATRRYCIPEHA-BQBZGAKWSA-N Gly-Glu-Glu Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SOEATRRYCIPEHA-BQBZGAKWSA-N 0.000 description 3
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 3
- LOEANKRDMMVOGZ-YUMQZZPRSA-N Gly-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O LOEANKRDMMVOGZ-YUMQZZPRSA-N 0.000 description 3
- ISSDODCYBOWWIP-GJZGRUSLSA-N Gly-Pro-Trp Chemical compound [H]NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O ISSDODCYBOWWIP-GJZGRUSLSA-N 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- IAYPZSHNZQHQNO-KKUMJFAQSA-N His-Ser-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC2=CN=CN2)N IAYPZSHNZQHQNO-KKUMJFAQSA-N 0.000 description 3
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 3
- 229930064664 L-arginine Natural products 0.000 description 3
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 3
- CHJKEDSZNSONPS-DCAQKATOSA-N Leu-Pro-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O CHJKEDSZNSONPS-DCAQKATOSA-N 0.000 description 3
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 3
- SNIPWBQKOPCJRG-CIUDSAMLSA-N Pro-Gln-Cys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O SNIPWBQKOPCJRG-CIUDSAMLSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- DTPARJBMONKGGC-IHPCNDPISA-N Trp-Cys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N DTPARJBMONKGGC-IHPCNDPISA-N 0.000 description 3
- UNUZEBFXGWVAOP-DZKIICNBSA-N Tyr-Glu-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UNUZEBFXGWVAOP-DZKIICNBSA-N 0.000 description 3
- -1 Value linear Chemical compound 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000005277 cation exchange chromatography Methods 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000009545 invasion Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 238000007747 plating Methods 0.000 description 3
- 229920002704 polyhistidine Polymers 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000009465 prokaryotic expression Effects 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 230000007480 spreading Effects 0.000 description 3
- 238000003892 spreading Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- HKZAAJSTFUZYTO-LURJTMIESA-N (2s)-2-[[2-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoic acid Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O HKZAAJSTFUZYTO-LURJTMIESA-N 0.000 description 2
- 102220542295 39S ribosomal protein L15, mitochondrial_R95E_mutation Human genes 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 101710153593 Albumin A Proteins 0.000 description 2
- WVNFNPGXYADPPO-BQBZGAKWSA-N Arg-Gly-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O WVNFNPGXYADPPO-BQBZGAKWSA-N 0.000 description 2
- YRTOMUMWSTUQAX-FXQIFTODSA-N Asn-Pro-Asp Chemical compound NC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O YRTOMUMWSTUQAX-FXQIFTODSA-N 0.000 description 2
- PORWNQWEEIOIRH-XHNCKOQMSA-N Cys-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CS)N)C(=O)O PORWNQWEEIOIRH-XHNCKOQMSA-N 0.000 description 2
- DZLQXIFVQFTFJY-BYPYZUCNSA-N Cys-Gly-Gly Chemical compound SC[C@H](N)C(=O)NCC(=O)NCC(O)=O DZLQXIFVQFTFJY-BYPYZUCNSA-N 0.000 description 2
- 241001269524 Dura Species 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102000010911 Enzyme Precursors Human genes 0.000 description 2
- 108010062466 Enzyme Precursors Proteins 0.000 description 2
- 238000011771 FVB mouse Methods 0.000 description 2
- OGNJZUXUTPQVBR-BQBZGAKWSA-N Glu-Gly-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O OGNJZUXUTPQVBR-BQBZGAKWSA-N 0.000 description 2
- GWCRIHNSVMOBEQ-BQBZGAKWSA-N Gly-Arg-Ser Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O GWCRIHNSVMOBEQ-BQBZGAKWSA-N 0.000 description 2
- ITZOBNKQDZEOCE-NHCYSSNCSA-N Gly-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN ITZOBNKQDZEOCE-NHCYSSNCSA-N 0.000 description 2
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 2
- IDNNYVGVSZMQTK-IHRRRGAJSA-N His-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N IDNNYVGVSZMQTK-IHRRRGAJSA-N 0.000 description 2
- FFKJUTZARGRVTH-KKUMJFAQSA-N His-Ser-Tyr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FFKJUTZARGRVTH-KKUMJFAQSA-N 0.000 description 2
- 101000898034 Homo sapiens Hepatocyte growth factor Proteins 0.000 description 2
- PDTMWFVVNZYWTR-NHCYSSNCSA-N Ile-Gly-Lys Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](CCCCN)C(O)=O PDTMWFVVNZYWTR-NHCYSSNCSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 125000002059 L-arginyl group Chemical class O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(=N[H])N([H])[H] 0.000 description 2
- FDBTVENULFNTAL-XQQFMLRXSA-N Leu-Val-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N FDBTVENULFNTAL-XQQFMLRXSA-N 0.000 description 2
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 2
- 208000001940 Massive Hepatic Necrosis Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- AJCRQOHDLCBHFA-SRVKXCTJSA-N Pro-His-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O AJCRQOHDLCBHFA-SRVKXCTJSA-N 0.000 description 2
- OHKFXGKHSJKKAL-NRPADANISA-N Ser-Glu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OHKFXGKHSJKKAL-NRPADANISA-N 0.000 description 2
- ZOPISOXXPQNOCO-SVSWQMSJSA-N Ser-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CO)N ZOPISOXXPQNOCO-SVSWQMSJSA-N 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- YDPFWRVQHFWBKI-GVXVVHGQSA-N Val-Glu-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N YDPFWRVQHFWBKI-GVXVVHGQSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000001261 affinity purification Methods 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 210000000270 basal cell Anatomy 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000006471 dimerization reaction Methods 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 108010028295 histidylhistidine Proteins 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 229960002725 isoflurane Drugs 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- 238000009940 knitting Methods 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 201000006417 multiple sclerosis Diseases 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000003566 phosphorylation assay Methods 0.000 description 2
- 230000036470 plasma concentration Effects 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000002028 premature Effects 0.000 description 2
- 108010004914 prolylarginine Proteins 0.000 description 2
- 239000012217 radiopharmaceutical Substances 0.000 description 2
- 229940121896 radiopharmaceutical Drugs 0.000 description 2
- 230000002799 radiopharmaceutical effect Effects 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 102220067450 rs754763002 Human genes 0.000 description 2
- 102220254288 rs767276648 Human genes 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 230000001960 triggered effect Effects 0.000 description 2
- 208000019553 vascular disease Diseases 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 210000004885 white matter Anatomy 0.000 description 2
- WFLOTYSKFUPZQB-UHFFFAOYSA-N 1,2-difluoroethene Chemical group FC=CF WFLOTYSKFUPZQB-UHFFFAOYSA-N 0.000 description 1
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 description 1
- ZCWPHDXKEDBCER-UHFFFAOYSA-N 2,5-diphenyl-2h-tetrazol-2-ium;bromide Chemical class [Br-].C1=CC=CC=C1C1=[NH+]N(C=2C=CC=CC=2)N=N1 ZCWPHDXKEDBCER-UHFFFAOYSA-N 0.000 description 1
- UWSONZCNXUSTKW-UHFFFAOYSA-N 4,5-Dimethylthiazole Chemical compound CC=1N=CSC=1C UWSONZCNXUSTKW-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- CFNMUZCFSDMZPQ-GHXNOFRVSA-N 7-[(z)-3-methyl-4-(4-methyl-5-oxo-2h-furan-2-yl)but-2-enoxy]chromen-2-one Chemical compound C=1C=C2C=CC(=O)OC2=CC=1OC/C=C(/C)CC1OC(=O)C(C)=C1 CFNMUZCFSDMZPQ-GHXNOFRVSA-N 0.000 description 1
- QDRGPQWIVZNJQD-CIUDSAMLSA-N Ala-Arg-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O QDRGPQWIVZNJQD-CIUDSAMLSA-N 0.000 description 1
- XEXJJJRVTFGWIC-FXQIFTODSA-N Ala-Asn-Arg Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XEXJJJRVTFGWIC-FXQIFTODSA-N 0.000 description 1
- GSCLWXDNIMNIJE-ZLUOBGJFSA-N Ala-Asp-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O GSCLWXDNIMNIJE-ZLUOBGJFSA-N 0.000 description 1
- OKIKVSXTXVVFDV-MMWGEVLESA-N Ala-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N OKIKVSXTXVVFDV-MMWGEVLESA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- GHNDBBVSWOWYII-LPEHRKFASA-N Arg-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O GHNDBBVSWOWYII-LPEHRKFASA-N 0.000 description 1
- ZTKHZAXGTFXUDD-VEVYYDQMSA-N Arg-Asn-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZTKHZAXGTFXUDD-VEVYYDQMSA-N 0.000 description 1
- GDVDRMUYICMNFJ-CIUDSAMLSA-N Arg-Cys-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(O)=O GDVDRMUYICMNFJ-CIUDSAMLSA-N 0.000 description 1
- PPPXVIBMLFWNSK-BQBZGAKWSA-N Arg-Gly-Cys Chemical compound C(C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N PPPXVIBMLFWNSK-BQBZGAKWSA-N 0.000 description 1
- KRQSPVKUISQQFS-FJXKBIBVSA-N Arg-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N KRQSPVKUISQQFS-FJXKBIBVSA-N 0.000 description 1
- FSNVAJOPUDVQAR-AVGNSLFASA-N Arg-Lys-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FSNVAJOPUDVQAR-AVGNSLFASA-N 0.000 description 1
- RIIVUOJDDQXHRV-SRVKXCTJSA-N Arg-Lys-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O RIIVUOJDDQXHRV-SRVKXCTJSA-N 0.000 description 1
- ZEBDYGZVMMKZNB-SRVKXCTJSA-N Arg-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCN=C(N)N)N ZEBDYGZVMMKZNB-SRVKXCTJSA-N 0.000 description 1
- DNLQVHBBMPZUGJ-BQBZGAKWSA-N Arg-Ser-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O DNLQVHBBMPZUGJ-BQBZGAKWSA-N 0.000 description 1
- ZUVDFJXRAICIAJ-BPUTZDHNSA-N Arg-Trp-Asp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CC(O)=O)C(O)=O)=CNC2=C1 ZUVDFJXRAICIAJ-BPUTZDHNSA-N 0.000 description 1
- XOZYYXMHMIEJET-XIRDDKMYSA-N Arg-Trp-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(O)=O XOZYYXMHMIEJET-XIRDDKMYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- JJGRJMKUOYXZRA-LPEHRKFASA-N Asn-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)N)N)C(=O)O JJGRJMKUOYXZRA-LPEHRKFASA-N 0.000 description 1
- XVAPVJNJGLWGCS-ACZMJKKPSA-N Asn-Glu-Asn Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N XVAPVJNJGLWGCS-ACZMJKKPSA-N 0.000 description 1
- UBKOVSLDWIHYSY-ACZMJKKPSA-N Asn-Glu-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O UBKOVSLDWIHYSY-ACZMJKKPSA-N 0.000 description 1
- OPEPUCYIGFEGSW-WDSKDSINSA-N Asn-Gly-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O OPEPUCYIGFEGSW-WDSKDSINSA-N 0.000 description 1
- PBSQFBAJKPLRJY-BYULHYEWSA-N Asn-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N PBSQFBAJKPLRJY-BYULHYEWSA-N 0.000 description 1
- NYGILGUOUOXGMJ-YUMQZZPRSA-N Asn-Lys-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O NYGILGUOUOXGMJ-YUMQZZPRSA-N 0.000 description 1
- RVHGJNGNKGDCPX-KKUMJFAQSA-N Asn-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N RVHGJNGNKGDCPX-KKUMJFAQSA-N 0.000 description 1
- WLVLIYYBPPONRJ-GCJQMDKQSA-N Asn-Thr-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O WLVLIYYBPPONRJ-GCJQMDKQSA-N 0.000 description 1
- YNQMEIJEWSHOEO-SRVKXCTJSA-N Asn-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O YNQMEIJEWSHOEO-SRVKXCTJSA-N 0.000 description 1
- RYEWQKQXRJCHIO-SRVKXCTJSA-N Asp-Asn-Tyr Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 RYEWQKQXRJCHIO-SRVKXCTJSA-N 0.000 description 1
- NAPNAGZWHQHZLG-ZLUOBGJFSA-N Asp-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N NAPNAGZWHQHZLG-ZLUOBGJFSA-N 0.000 description 1
- SMZCLQGDQMGESY-ACZMJKKPSA-N Asp-Gln-Cys Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N SMZCLQGDQMGESY-ACZMJKKPSA-N 0.000 description 1
- UBPMOJLRVMGTOQ-GARJFASQSA-N Asp-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC(=O)O)N)C(=O)O UBPMOJLRVMGTOQ-GARJFASQSA-N 0.000 description 1
- SPKCGKRUYKMDHP-GUDRVLHUSA-N Asp-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N SPKCGKRUYKMDHP-GUDRVLHUSA-N 0.000 description 1
- LIVXPXUVXFRWNY-CIUDSAMLSA-N Asp-Lys-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O LIVXPXUVXFRWNY-CIUDSAMLSA-N 0.000 description 1
- BWJZSLQJNBSUPM-FXQIFTODSA-N Asp-Pro-Asn Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O BWJZSLQJNBSUPM-FXQIFTODSA-N 0.000 description 1
- ZVGRHIRJLWBWGJ-ACZMJKKPSA-N Asp-Ser-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZVGRHIRJLWBWGJ-ACZMJKKPSA-N 0.000 description 1
- IWLZBRTUIVXZJD-OLHMAJIHSA-N Asp-Thr-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O IWLZBRTUIVXZJD-OLHMAJIHSA-N 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 description 1
- OYPRJOBELJOOCE-RKEGKUSMSA-N Calcium-47 Chemical compound [47Ca] OYPRJOBELJOOCE-RKEGKUSMSA-N 0.000 description 1
- OKTJSMMVPCPJKN-NJFSPNSNSA-N Carbon-14 Chemical compound [14C] OKTJSMMVPCPJKN-NJFSPNSNSA-N 0.000 description 1
- VYZAMTAEIAYCRO-BJUDXGSMSA-N Chromium-51 Chemical compound [51Cr] VYZAMTAEIAYCRO-BJUDXGSMSA-N 0.000 description 1
- DCJNIJAWIRPPBB-CIUDSAMLSA-N Cys-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CS)N DCJNIJAWIRPPBB-CIUDSAMLSA-N 0.000 description 1
- DZIGZIIJIGGANI-FXQIFTODSA-N Cys-Glu-Gln Chemical compound SC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O DZIGZIIJIGGANI-FXQIFTODSA-N 0.000 description 1
- UXUSHQYYQCZWET-WDSKDSINSA-N Cys-Glu-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O UXUSHQYYQCZWET-WDSKDSINSA-N 0.000 description 1
- XTHUKRLJRUVVBF-WHFBIAKZSA-N Cys-Gly-Ser Chemical compound SC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O XTHUKRLJRUVVBF-WHFBIAKZSA-N 0.000 description 1
- ZLHPWFSAUJEEAN-KBIXCLLPSA-N Cys-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CS)N ZLHPWFSAUJEEAN-KBIXCLLPSA-N 0.000 description 1
- OZHXXYOHPLLLMI-CIUDSAMLSA-N Cys-Lys-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OZHXXYOHPLLLMI-CIUDSAMLSA-N 0.000 description 1
- VXLXATVURDNDCG-CIUDSAMLSA-N Cys-Lys-Asp Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CS)N VXLXATVURDNDCG-CIUDSAMLSA-N 0.000 description 1
- GDNWBSFSHJVXKL-GUBZILKMSA-N Cys-Lys-Gln Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O GDNWBSFSHJVXKL-GUBZILKMSA-N 0.000 description 1
- GFMJUESGWILPEN-MELADBBJSA-N Cys-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CS)N)C(=O)O GFMJUESGWILPEN-MELADBBJSA-N 0.000 description 1
- NDNZRWUDUMTITL-FXQIFTODSA-N Cys-Ser-Val Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NDNZRWUDUMTITL-FXQIFTODSA-N 0.000 description 1
- YWEHYKGJWHPGPY-XGEHTFHBSA-N Cys-Thr-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CS)N)O YWEHYKGJWHPGPY-XGEHTFHBSA-N 0.000 description 1
- MJOYUXLETJMQGG-IHRRRGAJSA-N Cys-Tyr-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MJOYUXLETJMQGG-IHRRRGAJSA-N 0.000 description 1
- VXDXZGYXHIADHF-YJRXYDGGSA-N Cys-Tyr-Thr Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VXDXZGYXHIADHF-YJRXYDGGSA-N 0.000 description 1
- 229940122029 DNA synthesis inhibitor Drugs 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical class CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 206010063560 Excessive granulation tissue Diseases 0.000 description 1
- GYHNNYVSQQEPJS-OIOBTWANSA-N Gallium-67 Chemical compound [67Ga] GYHNNYVSQQEPJS-OIOBTWANSA-N 0.000 description 1
- GYHNNYVSQQEPJS-YPZZEJLDSA-N Gallium-68 Chemical compound [68Ga] GYHNNYVSQQEPJS-YPZZEJLDSA-N 0.000 description 1
- SSWAFVQFQWOJIJ-XIRDDKMYSA-N Gln-Arg-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N SSWAFVQFQWOJIJ-XIRDDKMYSA-N 0.000 description 1
- XIPZDANNDPMZGQ-WHFBIAKZSA-N Gln-Cys Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(O)=O XIPZDANNDPMZGQ-WHFBIAKZSA-N 0.000 description 1
- GFLNKSQHOBOMNM-AVGNSLFASA-N Gln-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCC(=O)N)N GFLNKSQHOBOMNM-AVGNSLFASA-N 0.000 description 1
- RSUVOPBMWMTVDI-XEGUGMAKSA-N Glu-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCC(O)=O)C)C(O)=O)=CNC2=C1 RSUVOPBMWMTVDI-XEGUGMAKSA-N 0.000 description 1
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 1
- XMPAXPSENRSOSV-RYUDHWBXSA-N Glu-Gly-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XMPAXPSENRSOSV-RYUDHWBXSA-N 0.000 description 1
- WVTIBGWZUMJBFY-GUBZILKMSA-N Glu-His-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O WVTIBGWZUMJBFY-GUBZILKMSA-N 0.000 description 1
- UDEPRBFQTWGLCW-CIUDSAMLSA-N Glu-Pro-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O UDEPRBFQTWGLCW-CIUDSAMLSA-N 0.000 description 1
- CAQXJMUDOLSBPF-SUSMZKCASA-N Glu-Thr-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAQXJMUDOLSBPF-SUSMZKCASA-N 0.000 description 1
- KCCNSVHJSMMGFS-NRPADANISA-N Glu-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N KCCNSVHJSMMGFS-NRPADANISA-N 0.000 description 1
- LZEUDRYSAZAJIO-AUTRQRHGSA-N Glu-Val-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LZEUDRYSAZAJIO-AUTRQRHGSA-N 0.000 description 1
- MFVQGXGQRIXBPK-WDSKDSINSA-N Gly-Ala-Glu Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O MFVQGXGQRIXBPK-WDSKDSINSA-N 0.000 description 1
- GGEJHJIXRBTJPD-BYPYZUCNSA-N Gly-Asn-Gly Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GGEJHJIXRBTJPD-BYPYZUCNSA-N 0.000 description 1
- JVWPPCWUDRJGAE-YUMQZZPRSA-N Gly-Asn-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JVWPPCWUDRJGAE-YUMQZZPRSA-N 0.000 description 1
- XRTDOIOIBMAXCT-NKWVEPMBSA-N Gly-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)CN)C(=O)O XRTDOIOIBMAXCT-NKWVEPMBSA-N 0.000 description 1
- QSTLUOIOYLYLLF-WDSKDSINSA-N Gly-Asp-Glu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QSTLUOIOYLYLLF-WDSKDSINSA-N 0.000 description 1
- PMNHJLASAAWELO-FOHZUACHSA-N Gly-Asp-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PMNHJLASAAWELO-FOHZUACHSA-N 0.000 description 1
- JMQFHZWESBGPFC-WDSKDSINSA-N Gly-Gln-Asp Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O JMQFHZWESBGPFC-WDSKDSINSA-N 0.000 description 1
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 1
- IDOGEHIWMJMAHT-BYPYZUCNSA-N Gly-Gly-Cys Chemical compound NCC(=O)NCC(=O)N[C@@H](CS)C(O)=O IDOGEHIWMJMAHT-BYPYZUCNSA-N 0.000 description 1
- ORXZVPZCPMKHNR-IUCAKERBSA-N Gly-His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CNC=N1 ORXZVPZCPMKHNR-IUCAKERBSA-N 0.000 description 1
- LRQXRHGQEVWGPV-NHCYSSNCSA-N Gly-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN LRQXRHGQEVWGPV-NHCYSSNCSA-N 0.000 description 1
- YSDLIYZLOTZZNP-UWVGGRQHSA-N Gly-Leu-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN YSDLIYZLOTZZNP-UWVGGRQHSA-N 0.000 description 1
- FGPLUIQCSKGLTI-WDSKDSINSA-N Gly-Ser-Glu Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O FGPLUIQCSKGLTI-WDSKDSINSA-N 0.000 description 1
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 1
- RJVZMGQMJOQIAX-GJZGRUSLSA-N Gly-Trp-Met Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCSC)C(O)=O RJVZMGQMJOQIAX-GJZGRUSLSA-N 0.000 description 1
- WRFOZIJRODPLIA-QWRGUYRKSA-N Gly-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)O WRFOZIJRODPLIA-QWRGUYRKSA-N 0.000 description 1
- GBYYQVBXFVDJPJ-WLTAIBSBSA-N Gly-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)CN)O GBYYQVBXFVDJPJ-WLTAIBSBSA-N 0.000 description 1
- ZVXMEWXHFBYJPI-LSJOCFKGSA-N Gly-Val-Ile Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZVXMEWXHFBYJPI-LSJOCFKGSA-N 0.000 description 1
- 102000009465 Growth Factor Receptors Human genes 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 238000012752 Hepatectomy Methods 0.000 description 1
- 102100022623 Hepatocyte growth factor receptor Human genes 0.000 description 1
- 101710184069 Hepatocyte growth factor receptor Proteins 0.000 description 1
- WCNXUTNLSRWWQN-DCAQKATOSA-N His-Asp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N WCNXUTNLSRWWQN-DCAQKATOSA-N 0.000 description 1
- JFFAPRNXXLRINI-NHCYSSNCSA-N His-Asp-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JFFAPRNXXLRINI-NHCYSSNCSA-N 0.000 description 1
- JWLWNCVBBSBCEM-NKIYYHGXSA-N His-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CN=CN1)N)O JWLWNCVBBSBCEM-NKIYYHGXSA-N 0.000 description 1
- PYNUBZSXKQKAHL-UWVGGRQHSA-N His-Gly-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O PYNUBZSXKQKAHL-UWVGGRQHSA-N 0.000 description 1
- DPQIPEAHIYMUEJ-IHRRRGAJSA-N His-Lys-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC1=CN=CN1)N DPQIPEAHIYMUEJ-IHRRRGAJSA-N 0.000 description 1
- DQZCEKQPSOBNMJ-NKIYYHGXSA-N His-Thr-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DQZCEKQPSOBNMJ-NKIYYHGXSA-N 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 description 1
- BOTVMTSMOUSDRW-GMOBBJLQSA-N Ile-Arg-Asn Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(N)=O)C(O)=O BOTVMTSMOUSDRW-GMOBBJLQSA-N 0.000 description 1
- AZEYWPUCOYXFOE-CYDGBPFRSA-N Ile-Arg-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](C(C)C)C(=O)O)N AZEYWPUCOYXFOE-CYDGBPFRSA-N 0.000 description 1
- LOXMWQOKYBGCHF-JBDRJPRFSA-N Ile-Cys-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O LOXMWQOKYBGCHF-JBDRJPRFSA-N 0.000 description 1
- FHCNLXMTQJNJNH-KBIXCLLPSA-N Ile-Cys-Gln Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)O FHCNLXMTQJNJNH-KBIXCLLPSA-N 0.000 description 1
- YKLOMBNBQUTJDT-HVTMNAMFSA-N Ile-His-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YKLOMBNBQUTJDT-HVTMNAMFSA-N 0.000 description 1
- CKRFDMPBSWYOBT-PPCPHDFISA-N Ile-Lys-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N CKRFDMPBSWYOBT-PPCPHDFISA-N 0.000 description 1
- KLJKJVXDHVUMMZ-KKPKCPPISA-N Ile-Phe-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N KLJKJVXDHVUMMZ-KKPKCPPISA-N 0.000 description 1
- VISRCHQHQCLODA-NAKRPEOUSA-N Ile-Pro-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)O)N VISRCHQHQCLODA-NAKRPEOUSA-N 0.000 description 1
- ZYVTXBXHIKGZMD-QSFUFRPTSA-N Ile-Val-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ZYVTXBXHIKGZMD-QSFUFRPTSA-N 0.000 description 1
- XEEYBQQBJWHFJM-AKLPVKDBSA-N Iron-59 Chemical compound [59Fe] XEEYBQQBJWHFJM-AKLPVKDBSA-N 0.000 description 1
- HASRFYOMVPJRPU-SRVKXCTJSA-N Leu-Arg-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HASRFYOMVPJRPU-SRVKXCTJSA-N 0.000 description 1
- CCQLQKZTXZBXTN-NHCYSSNCSA-N Leu-Gly-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CCQLQKZTXZBXTN-NHCYSSNCSA-N 0.000 description 1
- OMHLATXVNQSALM-FQUUOJAGSA-N Leu-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(C)C)N OMHLATXVNQSALM-FQUUOJAGSA-N 0.000 description 1
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 1
- RZXLZBIUTDQHJQ-SRVKXCTJSA-N Leu-Lys-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O RZXLZBIUTDQHJQ-SRVKXCTJSA-N 0.000 description 1
- RRVCZCNFXIFGRA-DCAQKATOSA-N Leu-Pro-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RRVCZCNFXIFGRA-DCAQKATOSA-N 0.000 description 1
- LINKCQUOMUDLKN-KATARQTJSA-N Leu-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(C)C)N)O LINKCQUOMUDLKN-KATARQTJSA-N 0.000 description 1
- HGLKOTPFWOMPOB-MEYUZBJRSA-N Leu-Thr-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HGLKOTPFWOMPOB-MEYUZBJRSA-N 0.000 description 1
- LFXSPAIBSZSTEM-PMVMPFDFSA-N Leu-Trp-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O)N LFXSPAIBSZSTEM-PMVMPFDFSA-N 0.000 description 1
- ARNIBBOXIAWUOP-MGHWNKPDSA-N Leu-Tyr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ARNIBBOXIAWUOP-MGHWNKPDSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- HGZHSNBZDOLMLH-DCAQKATOSA-N Lys-Asn-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N HGZHSNBZDOLMLH-DCAQKATOSA-N 0.000 description 1
- PXHCFKXNSBJSTQ-KKUMJFAQSA-N Lys-Asn-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N)O PXHCFKXNSBJSTQ-KKUMJFAQSA-N 0.000 description 1
- NTBFKPBULZGXQL-KKUMJFAQSA-N Lys-Asp-Tyr Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NTBFKPBULZGXQL-KKUMJFAQSA-N 0.000 description 1
- BYEBKXRNDLTGFW-CIUDSAMLSA-N Lys-Cys-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O BYEBKXRNDLTGFW-CIUDSAMLSA-N 0.000 description 1
- VEGLGAOVLFODGC-GUBZILKMSA-N Lys-Glu-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O VEGLGAOVLFODGC-GUBZILKMSA-N 0.000 description 1
- PBLLTSKBTAHDNA-KBPBESRZSA-N Lys-Gly-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PBLLTSKBTAHDNA-KBPBESRZSA-N 0.000 description 1
- WOEDRPCHKPSFDT-MXAVVETBSA-N Lys-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCCCN)N WOEDRPCHKPSFDT-MXAVVETBSA-N 0.000 description 1
- IUWMQCZOTYRXPL-ZPFDUUQYSA-N Lys-Ile-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O IUWMQCZOTYRXPL-ZPFDUUQYSA-N 0.000 description 1
- IVFUVMSKSFSFBT-NHCYSSNCSA-N Lys-Ile-Gly Chemical compound OC(=O)CNC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN IVFUVMSKSFSFBT-NHCYSSNCSA-N 0.000 description 1
- QOJDBRUCOXQSSK-AJNGGQMLSA-N Lys-Ile-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(O)=O QOJDBRUCOXQSSK-AJNGGQMLSA-N 0.000 description 1
- UQRZFMQQXXJTTF-AVGNSLFASA-N Lys-Lys-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O UQRZFMQQXXJTTF-AVGNSLFASA-N 0.000 description 1
- LNMKRJJLEFASGA-BZSNNMDCSA-N Lys-Phe-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LNMKRJJLEFASGA-BZSNNMDCSA-N 0.000 description 1
- RMOKGALPSPOYKE-KATARQTJSA-N Lys-Thr-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O RMOKGALPSPOYKE-KATARQTJSA-N 0.000 description 1
- OZVXDDFYCQOPFD-XQQFMLRXSA-N Lys-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N OZVXDDFYCQOPFD-XQQFMLRXSA-N 0.000 description 1
- WWWGMQHQSAUXBU-BQBZGAKWSA-N Met-Gly-Asn Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O WWWGMQHQSAUXBU-BQBZGAKWSA-N 0.000 description 1
- LHXFNWBNRBWMNV-DCAQKATOSA-N Met-Ser-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N LHXFNWBNRBWMNV-DCAQKATOSA-N 0.000 description 1
- WSPQHZOMTFFWGH-XGEHTFHBSA-N Met-Thr-Cys Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(O)=O WSPQHZOMTFFWGH-XGEHTFHBSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- QJGQUHMNIGDVPM-BJUDXGSMSA-N Nitrogen-13 Chemical compound [13N] QJGQUHMNIGDVPM-BJUDXGSMSA-N 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- RIYZXJVARWJLKS-KKUMJFAQSA-N Phe-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 RIYZXJVARWJLKS-KKUMJFAQSA-N 0.000 description 1
- OSBADCBXAMSPQD-YESZJQIVSA-N Phe-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N OSBADCBXAMSPQD-YESZJQIVSA-N 0.000 description 1
- PEFJUUYFEGBXFA-BZSNNMDCSA-N Phe-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 PEFJUUYFEGBXFA-BZSNNMDCSA-N 0.000 description 1
- VDTYRPWRWRCROL-UFYCRDLUSA-N Phe-Val-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 VDTYRPWRWRCROL-UFYCRDLUSA-N 0.000 description 1
- IEIFEYBAYFSRBQ-IHRRRGAJSA-N Phe-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N IEIFEYBAYFSRBQ-IHRRRGAJSA-N 0.000 description 1
- OAICVXFJPJFONN-OUBTZVSYSA-N Phosphorus-32 Chemical compound [32P] OAICVXFJPJFONN-OUBTZVSYSA-N 0.000 description 1
- 229920009405 Polyvinylidenefluoride (PVDF) Film Polymers 0.000 description 1
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 1
- ICTZKEXYDDZZFP-SRVKXCTJSA-N Pro-Arg-Pro Chemical compound N([C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(O)=O)C(=O)[C@@H]1CCCN1 ICTZKEXYDDZZFP-SRVKXCTJSA-N 0.000 description 1
- WECYCNFPGZLOOU-FXQIFTODSA-N Pro-Asn-Cys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O WECYCNFPGZLOOU-FXQIFTODSA-N 0.000 description 1
- SGCZFWSQERRKBD-BQBZGAKWSA-N Pro-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 SGCZFWSQERRKBD-BQBZGAKWSA-N 0.000 description 1
- PULPZRAHVFBVTO-DCAQKATOSA-N Pro-Glu-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PULPZRAHVFBVTO-DCAQKATOSA-N 0.000 description 1
- NMELOOXSGDRBRU-YUMQZZPRSA-N Pro-Glu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H]1CCCN1 NMELOOXSGDRBRU-YUMQZZPRSA-N 0.000 description 1
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 1
- BODDREDDDRZUCF-QTKMDUPCSA-N Pro-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@@H]2CCCN2)O BODDREDDDRZUCF-QTKMDUPCSA-N 0.000 description 1
- UREQLMJCKFLLHM-NAKRPEOUSA-N Pro-Ile-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UREQLMJCKFLLHM-NAKRPEOUSA-N 0.000 description 1
- SUENWIFTSTWUKD-AVGNSLFASA-N Pro-Leu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O SUENWIFTSTWUKD-AVGNSLFASA-N 0.000 description 1
- VGVCNKSUVSZEIE-IHRRRGAJSA-N Pro-Phe-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O VGVCNKSUVSZEIE-IHRRRGAJSA-N 0.000 description 1
- SPLBRAKYXGOFSO-UNQGMJICSA-N Pro-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@@H]2CCCN2)O SPLBRAKYXGOFSO-UNQGMJICSA-N 0.000 description 1
- PKHDJFHFMGQMPS-RCWTZXSCSA-N Pro-Thr-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PKHDJFHFMGQMPS-RCWTZXSCSA-N 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 229940123573 Protein synthesis inhibitor Drugs 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- IGLNJRXAVVLDKE-OIOBTWANSA-N Rubidium-82 Chemical compound [82Rb] IGLNJRXAVVLDKE-OIOBTWANSA-N 0.000 description 1
- BUGBHKTXTAQXES-AHCXROLUSA-N Selenium-75 Chemical compound [75Se] BUGBHKTXTAQXES-AHCXROLUSA-N 0.000 description 1
- 102000009203 Sema domains Human genes 0.000 description 1
- 108050000099 Sema domains Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- WTUJZHKANPDPIN-CIUDSAMLSA-N Ser-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N WTUJZHKANPDPIN-CIUDSAMLSA-N 0.000 description 1
- QEDMOZUJTGEIBF-FXQIFTODSA-N Ser-Arg-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O QEDMOZUJTGEIBF-FXQIFTODSA-N 0.000 description 1
- INCNPLPRPOYTJI-JBDRJPRFSA-N Ser-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N INCNPLPRPOYTJI-JBDRJPRFSA-N 0.000 description 1
- YPUSXTWURJANKF-KBIXCLLPSA-N Ser-Gln-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YPUSXTWURJANKF-KBIXCLLPSA-N 0.000 description 1
- KJMOINFQVCCSDX-XKBZYTNZSA-N Ser-Gln-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KJMOINFQVCCSDX-XKBZYTNZSA-N 0.000 description 1
- WSTIOCFMWXNOCX-YUMQZZPRSA-N Ser-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N WSTIOCFMWXNOCX-YUMQZZPRSA-N 0.000 description 1
- KDGARKCAKHBEDB-NKWVEPMBSA-N Ser-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CO)N)C(=O)O KDGARKCAKHBEDB-NKWVEPMBSA-N 0.000 description 1
- XXXAXOWMBOKTRN-XPUUQOCRSA-N Ser-Gly-Val Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXXAXOWMBOKTRN-XPUUQOCRSA-N 0.000 description 1
- HEUVHBXOVZONPU-BJDJZHNGSA-N Ser-Leu-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HEUVHBXOVZONPU-BJDJZHNGSA-N 0.000 description 1
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 1
- JUTGONBTALQWMK-NAKRPEOUSA-N Ser-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CO)N JUTGONBTALQWMK-NAKRPEOUSA-N 0.000 description 1
- VIIJCAQMJBHSJH-FXQIFTODSA-N Ser-Met-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O VIIJCAQMJBHSJH-FXQIFTODSA-N 0.000 description 1
- JJUNLJTUIKFPRF-BPUTZDHNSA-N Ser-Met-Trp Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CO)N JJUNLJTUIKFPRF-BPUTZDHNSA-N 0.000 description 1
- BVLGVLWFIZFEAH-BPUTZDHNSA-N Ser-Pro-Trp Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O BVLGVLWFIZFEAH-BPUTZDHNSA-N 0.000 description 1
- OLKICIBQRVSQMA-SRVKXCTJSA-N Ser-Ser-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OLKICIBQRVSQMA-SRVKXCTJSA-N 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- GKLVYJBZJHMRIY-OUBTZVSYSA-N Technetium-99 Chemical compound [99Tc] GKLVYJBZJHMRIY-OUBTZVSYSA-N 0.000 description 1
- CTONFVDJYCAMQM-IUKAMOBKSA-N Thr-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H]([C@@H](C)O)N CTONFVDJYCAMQM-IUKAMOBKSA-N 0.000 description 1
- CRZNCABIJLRFKZ-IUKAMOBKSA-N Thr-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N CRZNCABIJLRFKZ-IUKAMOBKSA-N 0.000 description 1
- YJCVECXVYHZOBK-KNZXXDILSA-N Thr-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H]([C@@H](C)O)N YJCVECXVYHZOBK-KNZXXDILSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- FLPZMPOZGYPBEN-PPCPHDFISA-N Thr-Leu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLPZMPOZGYPBEN-PPCPHDFISA-N 0.000 description 1
- CJXURNZYNHCYFD-WDCWCFNPSA-N Thr-Lys-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O CJXURNZYNHCYFD-WDCWCFNPSA-N 0.000 description 1
- MGJLBZFUXUGMML-VOAKCMCISA-N Thr-Lys-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MGJLBZFUXUGMML-VOAKCMCISA-N 0.000 description 1
- XSEPSRUDSPHMPX-KATARQTJSA-N Thr-Lys-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O XSEPSRUDSPHMPX-KATARQTJSA-N 0.000 description 1
- PCMDGXKXVMBIFP-VEVYYDQMSA-N Thr-Met-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMDGXKXVMBIFP-VEVYYDQMSA-N 0.000 description 1
- XKWABWFMQXMUMT-HJGDQZAQSA-N Thr-Pro-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XKWABWFMQXMUMT-HJGDQZAQSA-N 0.000 description 1
- GVMXJJAJLIEASL-ZJDVBMNYSA-N Thr-Pro-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O GVMXJJAJLIEASL-ZJDVBMNYSA-N 0.000 description 1
- STUAPCLEDMKXKL-LKXGYXEUSA-N Thr-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O STUAPCLEDMKXKL-LKXGYXEUSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- MHNHRNHJMXAVHZ-AAEUAGOBSA-N Trp-Asn-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N MHNHRNHJMXAVHZ-AAEUAGOBSA-N 0.000 description 1
- OFCKFBGRYHOKFP-IHPCNDPISA-N Trp-Asp-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N OFCKFBGRYHOKFP-IHPCNDPISA-N 0.000 description 1
- HXMJXDNSFVNSEH-IHPCNDPISA-N Trp-Cys-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N HXMJXDNSFVNSEH-IHPCNDPISA-N 0.000 description 1
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 description 1
- AKFLVKKWVZMFOT-IHRRRGAJSA-N Tyr-Arg-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O AKFLVKKWVZMFOT-IHRRRGAJSA-N 0.000 description 1
- JWHOIHCOHMZSAR-QWRGUYRKSA-N Tyr-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JWHOIHCOHMZSAR-QWRGUYRKSA-N 0.000 description 1
- SMLCYZYQFRTLCO-UWJYBYFXSA-N Tyr-Cys-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O SMLCYZYQFRTLCO-UWJYBYFXSA-N 0.000 description 1
- LOOCQRRBKZTPKO-AVGNSLFASA-N Tyr-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LOOCQRRBKZTPKO-AVGNSLFASA-N 0.000 description 1
- LTSIAOZUVISRAQ-QWRGUYRKSA-N Tyr-Gly-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)O LTSIAOZUVISRAQ-QWRGUYRKSA-N 0.000 description 1
- HIINQLBHPIQYHN-JTQLQIEISA-N Tyr-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 HIINQLBHPIQYHN-JTQLQIEISA-N 0.000 description 1
- ULHJJQYGMWONTD-HKUYNNGSSA-N Tyr-Gly-Trp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O ULHJJQYGMWONTD-HKUYNNGSSA-N 0.000 description 1
- XJPXTYLVMUZGNW-IHRRRGAJSA-N Tyr-Pro-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O XJPXTYLVMUZGNW-IHRRRGAJSA-N 0.000 description 1
- QHONGSVIVOFKAC-ULQDDVLXSA-N Tyr-Pro-His Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O QHONGSVIVOFKAC-ULQDDVLXSA-N 0.000 description 1
- REJBPZVUHYNMEN-LSJOCFKGSA-N Val-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N REJBPZVUHYNMEN-LSJOCFKGSA-N 0.000 description 1
- PVPAOIGJYHVWBT-KKHAAJSZSA-N Val-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N)O PVPAOIGJYHVWBT-KKHAAJSZSA-N 0.000 description 1
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 1
- FEXILLGKGGTLRI-NHCYSSNCSA-N Val-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N FEXILLGKGGTLRI-NHCYSSNCSA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- YTNGABPUXFEOGU-SRVKXCTJSA-N Val-Pro-Arg Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O YTNGABPUXFEOGU-SRVKXCTJSA-N 0.000 description 1
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 1
- NHXZRXLFOBFMDM-AVGNSLFASA-N Val-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C NHXZRXLFOBFMDM-AVGNSLFASA-N 0.000 description 1
- RYHUIHUOYRNNIE-NRPADANISA-N Val-Ser-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N RYHUIHUOYRNNIE-NRPADANISA-N 0.000 description 1
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 1
- LCHZBEUVGAVMKS-RHYQMDGZSA-N Val-Thr-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(O)=O LCHZBEUVGAVMKS-RHYQMDGZSA-N 0.000 description 1
- GUIYPEKUEMQBIK-JSGCOSHPSA-N Val-Tyr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(O)=O GUIYPEKUEMQBIK-JSGCOSHPSA-N 0.000 description 1
- VWQVUPCCIRVNHF-OUBTZVSYSA-N Yttrium-90 Chemical compound [90Y] VWQVUPCCIRVNHF-OUBTZVSYSA-N 0.000 description 1
- HZWXJJCSDBQVLF-UHFFFAOYSA-N acetoxysulfonic acid Chemical compound CC(=O)OS(O)(=O)=O HZWXJJCSDBQVLF-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000008275 binding mechanism Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- OKTJSMMVPCPJKN-BJUDXGSMSA-N carbon-11 Chemical compound [11C] OKTJSMMVPCPJKN-BJUDXGSMSA-N 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000005961 cardioprotection Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- GUTLYIVDDKVIGB-YPZZEJLDSA-N cobalt-57 Chemical compound [57Co] GUTLYIVDDKVIGB-YPZZEJLDSA-N 0.000 description 1
- GUTLYIVDDKVIGB-BJUDXGSMSA-N cobalt-58 Chemical compound [58Co] GUTLYIVDDKVIGB-BJUDXGSMSA-N 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 239000013632 covalent dimer Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- 108010054813 diprotin B Proteins 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- UYAHIZSMUZPPFV-NJFSPNSNSA-N erbium-169 Chemical compound [169Er] UYAHIZSMUZPPFV-NJFSPNSNSA-N 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 229960003699 evans blue Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229940006110 gallium-67 Drugs 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 1
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 1
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 1
- 108010079413 glycyl-prolyl-glutamic acid Proteins 0.000 description 1
- 108010008671 glycyl-tryptophyl-methionine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 230000004217 heart function Effects 0.000 description 1
- HLUCICHZHWJHLL-UHFFFAOYSA-N hematein Chemical compound C12=CC=C(O)C(O)=C2OCC2(O)C1=C1C=C(O)C(=O)C=C1C2 HLUCICHZHWJHLL-UHFFFAOYSA-N 0.000 description 1
- 239000013542 high molecular weight contaminant Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000010231 histologic analysis Methods 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 102000057308 human HGF Human genes 0.000 description 1
- 238000002675 image-guided surgery Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 229940055742 indium-111 Drugs 0.000 description 1
- APFVFJFRJDLVQX-AHCXROLUSA-N indium-111 Chemical compound [111In] APFVFJFRJDLVQX-AHCXROLUSA-N 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- XMBWDFGMSWQBCA-YPZZEJLDSA-N iodane Chemical compound [125IH] XMBWDFGMSWQBCA-YPZZEJLDSA-N 0.000 description 1
- 229940044173 iodine-125 Drugs 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 108010027338 isoleucylcysteine Proteins 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229910052743 krypton Inorganic materials 0.000 description 1
- DNNSSWSSYDEUBZ-UHFFFAOYSA-N krypton atom Chemical compound [Kr] DNNSSWSSYDEUBZ-UHFFFAOYSA-N 0.000 description 1
- 238000003475 lamination Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 108010056582 methionylglutamic acid Proteins 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000013425 morphometry Methods 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000033667 organ regeneration Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- QVGXLLKOCUKJST-BJUDXGSMSA-N oxygen-15 atom Chemical compound [15O] QVGXLLKOCUKJST-BJUDXGSMSA-N 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 229940097886 phosphorus 32 Drugs 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000000007 protein synthesis inhibitor Substances 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 229960005562 radium-223 Drugs 0.000 description 1
- HCWPIIXVSYCSAN-OIOBTWANSA-N radium-223 Chemical compound [223Ra] HCWPIIXVSYCSAN-OIOBTWANSA-N 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical class [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- KZUNJOHGWZRPMI-AKLPVKDBSA-N samarium-153 Chemical compound [153Sm] KZUNJOHGWZRPMI-AKLPVKDBSA-N 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000036560 skin regeneration Effects 0.000 description 1
- KEAYESYHFKHZAL-BJUDXGSMSA-N sodium-22 Chemical compound [22Na] KEAYESYHFKHZAL-BJUDXGSMSA-N 0.000 description 1
- KEAYESYHFKHZAL-OUBTZVSYSA-N sodium-24 Chemical compound [24Na] KEAYESYHFKHZAL-OUBTZVSYSA-N 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 229940006509 strontium-89 Drugs 0.000 description 1
- CIOAGBVUUVVLOB-OUBTZVSYSA-N strontium-89 Chemical compound [89Sr] CIOAGBVUUVVLOB-OUBTZVSYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229940056501 technetium 99m Drugs 0.000 description 1
- BKVIYDNLLOSFOA-OIOBTWANSA-N thallium-201 Chemical compound [201Tl] BKVIYDNLLOSFOA-OIOBTWANSA-N 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000012301 transgenic model Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
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
- C07K14/475—Growth factors; Growth regulators
- C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Abstract
本发明涉及来源于的HGF/SF的蛋白质,其能够诱导酪氨酸激酶受体MET的活化,以及所述蛋白质的用途,特别是用于促进组织再生。
Description
本发明涉及包含来自肝细胞生长因子/扩散因子(HGF/SF)的两个K1结构域的蛋白质。
肝细胞生长因子/扩散因子(HGF/SF)是具有复合结构域结构的分泌型90kDa蛋白质,其被合成为无活性前体,随后被蛋白水解转化为双链(α/β)活性物质(Nakamura,T.,Structure and function of hepatocyte growth factor.Prog Growth Factor Res 3,67-85(1991);Holmes等人,Insights into the structure/function of hepatocytegrowth factor/scatter factor from studies with individual domains.J MolBiol367,395-408(2007))。α链由N端结构域(N)和kringle结构域的四个拷贝(K1,K2,K3和K4)组成。β链是催化无活性的丝氨酸蛋白酶同源结构域(SPH)。已经在HGF/SF中鉴定了两个受体结合位点:位于α链的N末端区域的高亲和力位点和位于β链的低亲和力位点。
HGF/SF是一种有效的生长和运动因子,其被独立发现为肝促***原(肝细胞生长因子,HGF)(Miyazawa等人,Molecular cloning and sequence analysis of cDNA forhuman hepatocyte growth factor.Biochem Biophys Res Commun 163,967-973(1989);Nakamura等人,Purification and subunit structure of hepatocyte growth factorfrom rat platelets.FEBS Lett 224,311-316(1998);Zarnegar等人,Purification andbiological characterization of human hepatopoietin a,a polypeptide growthfactor for hepatocytes.Cancer Res 49,3314-3320(1989))和成纤维细胞衍生的上皮运动因子(分散因子,SF)(Stoker等人,Scatter factor is a fibroblast-derivedmodulator of epithelial cell mobility.Nature 327,239-242(1987);Gherardi等人,Purification of scatter factor,a fibroblast-derived basic protein thatmodulates epithelial interactions and movement.Proc Natl Acad Sci USA 86,5844-5848(1989))。由原癌基因编码的受体酪氨酸激酶MET随后被证明是HGF/SF的受体(Bottaro等人,Identification of the hepatocyte growth factor as the c-metproto-oncogene product.Science 251,802-804(1991))。
有趣的是,主要HGF/SF转录物编码两种可变剪接变体。第一种变体是由过早翻译终止引起的,并产生含有N结构域和HGF/SF的第一个Kringle结构域(K1)的NK1蛋白。NK1蛋白具有显著的激动剂活性,但需要硫酸乙酰肝素相互作用以诱导完全的MET活化。在结构上,NK1蛋白由两个球状结构域组成,在肝素存在下,所述结构域形成可能导致MET二聚化和活化的“头尾”同型二聚体(Chirgadze等人,Crystal structure of the NK1fragment ofHGF/SF suggests a novel mode for growth factor dimerization and receptorbinding.Nat Struct Biol 6,72-79(1999)。第二种剪接变体也是由过早终止产生的,其产生含有N结构域和前两个kringle结构域的NK2蛋白,NK2被认为是天然的MET拮抗剂。实际上,NK2维持其MET结合能力,但由于其构象特性,缺乏激活MET的能力。然而,基于结构的靶向突变允许NK2通过以接近NK1的构象重新定位K1结构域而从MET拮抗剂有效切换至激动剂。
除了提出统一和收敛的相互作用模型的许多尝试之外,HGF/SF的MET结合机制仍不清楚和有争议。特别地,在与可溶性MET细胞外结构域的复合物中没有NK1的晶体结构。HGF/SF是二价配体,其含有用于MET的分别位于α-链(N和/或K1结构域)的N-末端区域和β-链(SPH结构域)中的高和低亲和力结合位点。在溶液中HGF/SF与MET胞外域的结合产生具有2:2化学计量的复合物(Gherardi等人,Structural basis of hepatocyte growthfactor/scatter factor and met signalling.Proc Natl Acad Sci USA 103,4046-4051(2006))。SPH结构域以定义明确的界面结合MET。然而,NK1结合位点在MET上的定位仍不清楚,并且确切的HGF/SF-MET相互作用模型仍然存在争议(Holmes等人,Insights into thestructure/function of hepatocyte growth factor/scatter factor from studieswith individual domains.J Mol Biol 367,395-408(2007);Stamos等人,Crystalstructure of the HGF beta-chain in complex with the sema domain of the metreceptor.The EMBO Journal 23,2325-2335(2004);Merkulova-Rainon等人,The N-terminal domain of hepatocyte growth factor inhibits the angiogenic behaviorof endothelial cells independently from binding to the c-Met-receptor.J BiolChem 278,37400-37408(2003))。
HGF/SF和MET在发育和组织/器官再生中发挥重要的生理作用。特别地,HGF/SF-MET是肝切除术(Huh等人,Hepatocyte growth factor/c-met signaling pathway isrequired for efficient liver regeneration and repair.Proc Natl Acad Sci USA101,4447-4482(2004);Borowiak等人,Met provides essential signals for liverregeneration.Proc Natl Acad Sci USA 101,10608-10613(2004))和皮肤伤口(Chmielowiec et al.,C-met is essential for wound healing in the skin.J CellBiol 177,151-162(2007))后的肝脏和皮肤再生所必需的。HGF/SF进一步保护心脏和骨骼肌免受实验损伤(Urbanek等人,Cardiac stem cells possess growth factor-receptorsystems that after activation regenerate the infarcted myocardium,improvingventricular function and long-term survival.Circ Res 97,663-673(2005)),延迟运动神经元疾病的转基因模型(Sun等人,Overexpression of HGF retards diseaseprogression and prolongs life span in a transgenic mouse model of ALS.JNeurosci 22,6537-6548(2002))和多发性硬化症的免疫学模型(Bai等人,Hepatocytegrowth factor mediates mesenchymal stem cell-induced recovery in multiplesclerosis models.Nature neuroscience 15,862-870(2012))的进展。总之,这些研究突出了HGF/SF在再生医学中的巨大潜力,这一概念得到了许多临床前和最近的临床研究的支持。
特别地,许多研究集中在HGF/SF激动剂合成以允许组织再生,特别是用于在肝切除术或涉及糖尿病疾病的病变之后的肝再生。
由于HGF/SF-MET相互作用的现有知识不能实现HGF/SF-MET激动剂的合理设计,HGF/SF的有用性已经使用天然HGF/SF、基因递送方法和基于NK1的MET激动剂建立。
然而,天然HGF/SF是具有有限组织扩散的蛋白质,这反映其作为局部作用组织成形素的作用(Birchmeier等人,Met,metastasis,mobility and more.Nat Rev Mol CellBiol 4,915-925(2003);Ross等人,Protein Engineered Variants of HepatocyteGrowth Factor/Scatter Factor Promote Proliferation of Primary HumanHepatocytes and in Rodent Liver.Gastroenterology 142,897-906(2012))。实际上,在局部或全身给药后,HGF/SF被存在于细胞外基质中的硫酸乙酰肝素固定,导致在更远的组织中对MET受体的扩散严重降低(Roos等人,Induction of liver growth in normal miceby infusion of hepatocyte growth factor/scatter factor.The American Journalof Physiology 268,G380-386(1995);Hartmann等人,Engineered mutants of HGF/SFwith reduced binding to heparan sulfate proteoglycans,decreased clearance andenhanced activity in vivo.Curr Biol 8,125-134(1998))。此外,由于其复杂的多结构域结构,天然HGF/SH也难以生产,并且成本非常高。
基因递送方法,包括肌内注射编码HGF/SF的裸DNA,解决与使用天然HGF/SF作为蛋白质治疗相关的几个问题(例如生产HGF/SF蛋白质的成本)。HGF/SF DNA的临床试验目前在糖尿病周围神经病变患者和肌萎缩性侧索硬化患者中进行。这些研究的结果令人期待,但是目前的基因递送方法在基因产物的稳定治疗水平的实现以及由于有限扩散而对特定组织结构域和器官的相对可用性方面仍然存在限制。例如,这样的基因递送方法基于质粒递送***(专利申请WO 2009/093880,WO 2009/125986和WO 2013/065913)或基于腺病毒的递送***(Yang et al,.Improvement of heart function in postinfarct heart failureswine models after hepatocyte growth factor gene transfer:comparison of low-,medium-and high-doses groups.Mol Biol Rep 37,2075-2081(2010))。
目前可用的基于NK1的MET激动剂,例如1K1(即NK1突变体)具有强的激动活性并且提供优于HGF/SF的优势(Lietha等人,Crystal structures of NK1-heparin complexesreveal the basis for NK1activity and enable engineering of potent agonists ofthe MET receptor.The EMBO journal 20,5543-5555(2001)和专利US 7,179,786)。不同于天然HGF/SF,这种NK1突变体可以在异源表达***中有效产生,在生理缓冲液中是稳定的,因此在施用时可以完全控制剂量和血浆浓度。然而,NK1的潜在限制是其对于避免组织扩散的硫酸乙酰肝素的强残留亲和力。
因此,需要具有改善的稳定性、改善的保质期、最佳生物利用度并且可以以低成本生产和容易施用的有效的MET激动剂。
本发明的目的之一是提供能够诱导酪氨酸激酶受体MET活化的蛋白质。
本发明的另一个目的还是提供含有所述蛋白质的组合物。
本发明的另一个目的还涉及所述蛋白质的用途,特别是用于诊断和治疗应用。
本发明涉及含有两个肽结构域的蛋白质,所述肽结构域分别命名为K1a和K1b,所述肽结构域K1a和K1b各自包含肝细胞生长因子/扩散因子(HGF/SF)的K1肽结构域(Kringle1),
所述K1肽结构域由与SEQ ID NO:1具有至少80%同一性,优选至少90%同一性的序列组成,
所述蛋白质能够诱导酪氨酸激酶受体MET的活化。
本申请基于由发明人发现的意想不到的双方面观察:K1结构域构成有效MET激动剂的结构单元,并且可以改造具有有效MET激动剂活性的K1二聚体。
在本发明中,表述“K1a”和“K1b”全部是指包含HGF/SF的K1结构域的肽序列。
本发明的蛋白质是HGF/SF的K1结构域的共价二聚体,即两个K1结构域通过氨基酸链彼此共价连接。本发明的蛋白质可以用“K1K1”表示。
特别地,本发明涉及含有两个肽结构域的蛋白质,所述肽结构域分别命名为K1a和K1b,所述肽结构域K1a和K1b各自包含肝细胞生长因子/扩散因子(HGF/SF)的K1肽结构域(Kringle 1),
所述K1肽结构域由与SEQ ID NO:1具有至少80%同一性,优选至少90%同一性的序列组成,
所述蛋白质能够诱导酪氨酸激酶受体MET的活化,
条件是所述蛋白质不包含HGF/SF的N末端结构域。
本发明的蛋白质具有许多技术和经济上的优势。
最重要的技术优势是本发明的蛋白质具有强的MET激动活性。因此,该蛋白质能够在各种体外和体内测定中活化MET受体和/或诱导与MET活化相关的任何表型。
如果蛋白质能够:
-结合MET受体,
-活化细胞中的MET磷酸化和下游信号传导,
-诱导至少一种细胞表型,如存活、增殖、形态发生和/或迁移,
则该蛋白质具有MET激动活性。
这些标准的验证可以使用蛋白质-蛋白质相互作用测试(例如SPR(表面等离子体共振),AlphaScreen,下拉技术或凝胶过滤色谱法),磷酸化测试(如蛋白质印迹,ELISA或AlphaScreen)和表型测试(如扩散,MTT测定或MatrigelTM诱导的形态发生)来显示。
例如,细胞中的MET活化和下游信号传导可以通过蛋白质印迹法在体外进行分析,并通过均质时间分辨荧光(HTRF)方法进行定量。在体内,还可以对局部血管生成和保护小鼠免受Fas诱发的暴发性肝炎进行分析。
另一个技术优势是可以在完全控制剂量和/或血浆浓度的情况下施用本发明的蛋白质。
此外,天然HGF/SF的主要缺点之一是其强烈结合细胞外基质中的硫酸乙酰肝素。这严重限制了分子扩散到更远的位置。相反,本发明的蛋白质缺少高亲和力的硫酸乙酰肝素位点。
因此,与HGF/SF相反,该蛋白质不被细胞外基质的硫酸乙酰肝素链固定。因此,当注射到患者体内时,本发明的蛋白质可以从注射区扩散到远端组织中的MET受体,而天然HGF/SF不能。这通过小鼠的体内实验来证明。然而,在kringle结构域中低亲和力肝素结合位点的存在允许通过肝素-琼脂糖亲和层析有效纯化。
一些经济上的优势是本发明的蛋白质可以容易地以低成本大量生产。实际上,该蛋白质可以在用作表达***的宿主细胞例如细菌或酵母中重组产生。
在一个实施方案中,本发明涉及含有两个70-100个氨基酸的肽结构域的蛋白质,所述肽结构域分别命名为K1a和K1b,所述肽结构域K1a和K1b各自包含肝细胞生长因子/扩散因子(HGF/SF)的K1肽结构域(Kringle 1),
所述K1肽结构域由与SEQ ID NO:1具有至少80%同一性,优选至少90%同一性的序列组成,
所述蛋白质能够诱导酪氨酸激酶受体MET的活化。
在一个实施方案中,本发明涉及含有两个70-100个氨基酸的肽结构域的蛋白质,所述肽结构域分别命名为K1a和K1b,所述肽结构域K1a和K1b各自包含肝细胞生长因子/扩散因子(HGF/SF)的K1肽结构域(Kringle 1),
所述K1肽结构域由与SEQ ID NO:2具有至少80%同一性,优选至少90%同一性的序列组成,
所述蛋白质能够诱导酪氨酸激酶受体MET的活化。
在一个实施方案中,本发明涉及含有两个肽结构域的蛋白质,所述肽结构域分别命名为K1a和K1b,所述肽结构域K1a和K1b各自包含与SEQ ID NO:1具有至少80%同一性,优选至少90%的同一性的70-100个氨基酸的序列或由其组成,
所述蛋白质能够诱导酪氨酸激酶受体MET的活化。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中所述肽结构域K1a和K1b的大小各自为至少70个氨基酸,优选至少74个氨基酸,更优选至少79个氨基酸。
特别地,K1a和/或K1b的大小为70-100个氨基酸。这样的K1肽结构域可以由70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100个氨基酸组成。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中肽结构域K1a和K1b是相同的。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中肽结构域K1a和K1b彼此不同。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中肽结构域K1a和K1b各自由氨基酸序列SEQ ID NO:1组成。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中肽结构域K1a和K1b各自由氨基酸序列SEQ ID NO:2组成。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中肽结构域K1a和K1b各自由选自氨基酸序列SEQ ID NO:1和SEQ ID NO:2的氨基酸序列组成。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中所述肽结构域K1a和/或K1b包含相对于SEQ ID NO:1的至少一个氨基酸添加、缺失或取代,
K1a和K1b的K1肽结构域由与SEQ ID NO:1具有至少80%同一性的序列组成。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中所述肽结构域K1a和/或K1b包含相对于SEQ ID NO:1的位置5上的氨基酸K和/或位置7上的氨基酸R的至少一种取代,
K1a和K1b的K1肽结构域由与SEQ ID NO:1具有至少80%同一性的序列组成。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中所述肽结构域K1a和/或K1b包含相对于SEQ ID NO:1的选自K5E和R7E的至少一种取代,
K1a和K1b的K1肽结构域由与SEQ ID NO:1具有至少80%同一性的序列组成。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中所述肽结构域K1a和K1b各自包含相对于SEQ ID NO:1的取代K5E和R7E,
每个K1肽结构域由与SEQ ID NO:1具有至少80%同一性的序列组成。
SEQ ID NO:1对应于人K1结构域的序列,即由SEQ ID NO:12表示的HGF/SF的位置128的氨基酸至氨基酸位置206的区域。
SEQ ID NO:1具有79个氨基酸的大小,并且由两个半胱氨酸侧接。
SEQ ID NO:2对应于缺失5个氨基酸的人K1结构域的变体。
表1.HGF/SF和K1结构域的序列。
由SEQ ID NO:2表示的人K1结构域的变体由于缺失5个连续氨基酸:SFLPS而不同于SEQ ID NO:1所示的人K1结构域。
K1a和K1b可以由氨基酸序列SEQ ID NO:1或由与SEQ ID NO:1具有至少80%,81%,82%,83%,84%,85%,86%,87%,88%89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%同一性的氨基酸序列组成组成。
本发明定义的两个肽序列之间的“百分比同一性”是通过比较通过比较窗口最佳对比的两个序列来确定的。因此,比较窗口中的氨基酸序列可以包含相对于参考序列(其不包含添加或缺失)的添加或缺失(例如“间隙”),以便获得两个序列之间的最佳比对。
通过确定两个比较序列中的相同氨基酸残基的位置数,并将该数除以比较窗口中的位置总数并将结果乘以一百,得到两个氨基酸序列彼此的百分比同一性。
百分比同一性可以在整个氨基酸序列上或选定的结构域上,优选在整个氨基酸序列上测定。对于局部比对,Smith-Waterman算法特别有用(Smith T F,Waterman M S(1981)J.Mol.Biol.147(1);195-7)。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中肽结构域K1a和K1b各自由选自SEQ ID NO:3和SEQ ID NO:4的核酸序列编码。
SEQ ID NO:3对应于编码氨基酸序列SEQ ID NO:1的核酸序列。
SEQ ID NO:4对应于编码氨基酸序列SEQ ID NO:2的核酸序列。
表2.编码K1结构域的核酸序列。
在一个实施方案中,本发明涉及如上定义的蛋白质,所述蛋白质包含连接K1a和K1b的肽接头。
本发明涉及由两个肽结构域组成的蛋白质,所述肽结构域分别命名为K1a和K1b,通过肽接头相互连接,
所述肽结构域K1a和K1b各自包含肝细胞生长因子/扩散因子(HGF/SF)的K1肽结构域(Kringle 1),
所述K1肽结构域由与SEQ ID NO:1具有至少80%同一性,优选至少90%同一性的序列组成,
所述蛋白质能够诱导酪氨酸激酶受体MET的活化。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中所述肽接头由1至50个氨基酸,优选10至20个氨基酸构成。
特别地,接头的大小是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47、48、49或50个氨基酸。
在一个实施方案中,接头的大小为4个氨基酸。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中所述肽接头包含氨基酸序列SEQ ID NO:5(SEVE)或由氨基酸序列SEQ ID NO:5(SEVE)组成。
肽接头SEQ ID NO:5可以由核酸序列SEQ ID NO:6(TCAGAAGTTGAA)编码。
在一个实施方案中,本发明涉及包含氨基酸序列SEQ ID NO:7或与SEQ ID NO:7具有至少80%,优选90%同一性的氨基酸序列或由其组成的蛋白质。
在一个实施方案中,本发明涉及包含氨基酸序列SEQ ID NO:8或与SEQ ID NO:8具有至少80%,优选90%同一性的氨基酸序列或由其组成的蛋白质。
表3 K1K1蛋白的序列。
SEQ ID NO:7从N末端至C末端由SEQ ID NO:1,SEQ ID NO:6和SEQ ID NO:1构成。
SEQ ID NO:8从N末端至C末端由SEQ ID NO:2,SEQ ID NO:6和SEQ ID NO:2构成。
在一个实施方案中,本发明的蛋白质包含可切割标签,例如其N-端和/或C端最末端的多组氨酸标签。这种多组氨酸标签可用于允许标记蛋白质的亲和纯化。
在一个实施方案中,本发明涉及包含氨基酸序列SEQ ID NO:9或由氨基酸序列SEQID NO:9组成的蛋白质。
表4.C末端标记的K1K1蛋白的序列。
表5.K1K1变体。
SEQ ID NO:13对应于没有组氨酸标签的K1K1蛋白质。
SEQ ID NO:14对应于本发明的K1K1蛋白质,其中SEVE接头已被作为柔性接头的17个氨基酸序列(GGGGSLVPRGSGGGGS,SEQ ID NO:17)替代。该加长的接头包含允许分离两个K1结构域的凝血酶切割位点(LVPRGS,SEQ ID NO:18)。
SEQ ID NO:15对应于本发明的K1K1蛋白,其中SEVE接头已经被不存在任何结构约束的GS接头(GSGGS,SEQ ID NO:19)替代。
SEQ ID NO:16对应于本发明的K1K1蛋白,其中引入以下突变:K10E,R12E,K93E和R95E。K10E和R12E突变是第一个kringle结构域的一部分,K93E和R95E是第二个kringle结构域的一部分。靶向的K10、R12、K93和R95残基是与肝素相互作用的K1结构域的带正电荷片段的一部分。由于预期该构建体对肝素具有降低的亲和力,因此在C-末端添加六组氨酸标签,以便通过镍亲和层析纯化。
特别地,本发明涉及一种蛋白质,其包含或由以下组成:
-选自SEQ ID NO:9,SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15和SEQ ID NO:16的氨基酸序列,或
-与选自SEQ ID NO:9,SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15和SEQ ID NO:16的序列具有至少80%,优选90%同一性的氨基酸序列。
特别地,本发明的蛋白质包含与选自SEQ ID NO:9,SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15和SEQ ID NO:16的一个序列具有至少80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%同一性的氨基酸序列或由其组成。
特别地,本发明涉及包含选自SEQ ID NO:9,SEQ ID NO:13,SEQ ID NO:14,SEQ IDNO:15和SEQ ID NO:16的氨基酸序列或由其组成的蛋白质。
在一个实施方案中,本发明涉及如上定义的蛋白质,所述蛋白质是合成或重组蛋白质。
在本发明中,“合成蛋白质”是使用经典有机化学方法如液相或固相合成法合成的蛋白质。
在本发明中,“重组蛋白”是由遗传工程产生的蛋白质。可以使用经典的分子生物学技术将遗传构建体***载体中并在宿主细胞例如细菌或酵母中表达,以获得重组蛋白。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中酪氨酸激酶受体MET的活化是不依赖于硫酸乙酰肝素的。
在体内条件下,HGF/SF通过存在于细胞外基质中的硫酸乙酰肝素链固定,导致严重减少的扩散和/或组织分布。
本发明的蛋白质缺少高亲和力的硫酸乙酰肝素结合位点(N结构域),因此能够扩散到远端组织中的MET受体。
在一个实施方案中,本发明涉及如上定义的蛋白质,其中所述蛋白质能够以解离常数KD≤200nM,优选≤100nM,更优选≤10nM结合酪氨酸激酶受体MET。
特别地,所述蛋白质能够以解离常数KD≤200nM,≤150nM,≤100nM,≤90nM,≤80nM,≤70nM,≤60nM,≤50nM,≤40nM,≤30nM,≤10nM或≤5nM结合酪氨酸激酶受体MET。
在另一方面,本发明还涉及获得如上所定义的包含至少两个K1肽结构域的蛋白质的方法,包括以下步骤:
-在表达载体中***编码包含至少两个K1肽结构域,优选两个K1肽结构域的重组蛋白的核酸序列,
-将所述载体克隆在宿主细胞中,并表达所述重组蛋白,
-提取和纯化所述重组蛋白,所述重组蛋白是包含至少两个K1结构域的蛋白质。
另一方面,本发明涉及编码本发明蛋白的核酸分子,所述核酸优选由核酸序列SEQID NO:10组成。
表6.编码C末端标记的K1K1蛋白的核酸。
核酸序列SEQ ID NO:10编码氨基酸序列SEQ ID NO:9。
另一方面,本发明涉及含有如上定义的核酸分子的表达载体,所述载体优选包含核酸序列SEQ ID NO:11或由核酸序列SEQ ID NO:11组成。
表7.编码K1K1蛋白的表达载体。
另一方面,本发明涉及含有上述表达载体的宿主细胞,所述宿主细胞优选选自酵母细胞和细菌细胞。
在另一方面,本发明还涉及一种组合物,其包含:
-如上定义的蛋白质,或
-编码所述蛋白质的核酸分子,或
-含有所述核酸分子的表达载体,或
-含有所述表达载体的宿主细胞。
另一方面,本发明还涉及用于体内诊断方法的如上定义的蛋白质。
由于其结合MET的能力,本发明的蛋白质代表了用于诊断方法的有价值的工具,特别是涉及HGF/SF和MET分子表达的病状。
在一个实施方案中,本发明涉及如上定义的蛋白质,其用于诊断病状的方法,所述病状选自:上皮器官疾病,包括急性和慢性肝脏疾病、急性和慢性肾脏疾病、慢性肺部疾病和慢性皮肤伤口,中枢神经***疾病,包括神经元疾病和硬化,缺血性心脏病,外周血管疾病,糖尿病和相关并发症如周围神经病变。
在一个实施方案中,本发明涉及如上定义的蛋白质,其用于选自以下的病状的体内诊断方法:癌症,上皮器官疾病,包括急性和慢性肝脏疾病、急性和慢性肾脏疾病、慢性肺部疾病和慢性皮肤伤口,中枢神经***疾病,包括神经元疾病和硬化,缺血性心脏病,外周血管疾病,糖尿病和相关并发症如周围神经病变。
在一个实施方案中,本发明涉及用于体内诊断方法的如上所定义的蛋白质,其中所述癌症是表达酪氨酸激酶受体MET的肿瘤。
另一方面,本发明涉及用于医学成像的如上所定义的蛋白质。
另一方面,本发明涉及用于体内成像的如上定义的蛋白质。
在一个实施方案中,本发明涉及用于医学成像的如上所定义的蛋白质,其中所述蛋白质允许检测和/或跟踪药物和/或成像剂。
特别地,本发明的蛋白质可用于图像引导手术。手术前和术中成像被用于帮助外科医生仔细定位外科手术工具,以及指导完全去除特定组织。荧光(IR/NIR)探针可用于操作中的实时成像。
在另一方面,本发明涉及如上定义的蛋白质作为体外诊断工具的用途。
在一个实施方案中,本发明涉及如上定义的蛋白质用于病状的体外诊断的用途,所述病状选自:癌症,上皮器官疾病,包括急性和慢性肝脏疾病、急性和慢性肾脏疾病、慢性肺部疾病和慢性皮肤伤口,中枢神经***疾病,包括神经元疾病和硬化,缺血性心脏病,外周血管疾病,糖尿病和相关并发症如周围神经病变。
在一个实施方案中,本发明涉及蛋白质用于如上所述的病状的体外诊断的用途,其中所述癌症是表达酪氨酸激酶受体MET的肿瘤。
在另一方面,本发明涉及本发明的蛋白质用于体外或离体成像的用途。
在诊断方法和医学影像中,蛋白质可以通过剂量(例如使用活组织检查)或图片(从诸如PET扫描或IRM的技术获得)在生物样品中进行检测和定量。
实际上,本发明的蛋白质可以用标记物标记,并允许MET受体的检测,定位和定量。
例如,蛋白质可用放射性药物示踪剂或荧光示踪剂进行标记。
这种放射性药物示踪剂包括但不限于钙-47,碳-11,碳-14,铬-51,钴-57,钴-58,铒-169,氟-18,镓-67,镓-68,氢-3,铟-111,碘-123,碘-125,碘-131,铁-59,氪-81m,氮-13,氧-15,磷-32,镭-223,铷-82,钐-153,硒-75,钠-22,钠-24,锶-89,锝-99m,铊-201,氙-133和钇-90。
荧光示踪剂包括但不限于荧光染料(如若丹明衍生物,香豆素衍生物,荧光素衍生物,...)或荧光蛋白(例如GFP(绿色),YFP(黄色),RFP(红色)或基于植物色素的近红外荧光蛋白(iRFP)。
特别地,红外(IR)和近红外(NIR)染料和荧光蛋白由于增加的穿透和减少的自发荧光而是用于体内成像的优选示踪剂。
另一方面,本发明涉及一种用于体内诊断病状的方法,包括向患者施用如上定义的蛋白质的步骤,所述病状选自:癌症,上皮器官疾病,包括急性和慢性肝脏疾病、急性和慢性肾脏疾病、慢性肺部疾病和慢性皮肤伤口,中枢神经***疾病,包括神经元疾病和硬化,缺血性心脏病,外周血管疾病,糖尿病和相关并发症如周围神经病变。
另一方面,本发明涉及一种用于医学成像的方法,其包括向患者施用如上定义的蛋白质的步骤。
在一个实施方案中,本发明涉及用于医学成像的方法,其中所述蛋白质允许检测酪氨酸激酶受体MET。
在一个实施方案中,本发明涉及用于医学成像的方法,其中所述蛋白质允许抗体的预靶向。
实际上,本发明的蛋白质可以与识别示踪剂的特异性表位的抗体连接。
另一方面,本发明还涉及药物组合物,其包含:
-如上所定义的蛋白质
-编码所述蛋白质的核酸分子,或
-含有所述核酸分子的表达载体,或
-含有所述表达载体的宿主细胞,和
药学上可接受的载体。
另一方面,本发明还涉及用作药物的如上所定义的蛋白质。
在一个实施方案中,本发明涉及如上定义的蛋白质,其用于通过促进细胞存活或组织再生来治疗组织损伤。
在一个实施方案中,本发明涉及如上定义的蛋白质,用于治疗选自以下的病状:上皮器官疾病,包括急性和慢性肝脏疾病、急性和慢性肾脏疾病、慢性肺部疾病和慢性皮肤伤口,中枢神经***疾病,包括神经元疾病和硬化,缺血性心脏病,外周血管疾病,糖尿病和相关并发症如周围神经病变。
在一个实施方案中,本发明涉及如上定义的蛋白质,其用于治疗组织损伤或用于治疗如上所定义的病状,所述蛋白质可以以约1mg/kg至约1000mg/kg,优选约10mg/kg至约100mg/kg的剂量施用。
在一个实施方案中,本发明涉及如上定义的蛋白质,其用于治疗组织损伤或用于治疗如上所定义的病状,所述蛋白质以易于通过口服或静脉途径施用的形式以1mg至1000mg,特别是10mg至1000mg,特别是100至1000mg的单位剂量使用。
在另一方面,本发明还涉及如上定义的蛋白质在体内、离体或体外条件下促进血管发生的用途。
由于其有效的MET激动活性,本发明的蛋白质可用于了解MET与HGF/SF之间相互作用的机制。
另一方面,本发明涉及如上定义的蛋白质作为体外研究工具的用途。
另一方面,本发明还涉及如上定义的蛋白质和酪氨酸激酶受体MET之间的分子复合物,所述蛋白质与所述酪氨酸激酶受体MET通过至少两个K1结构域复合。
以下附图和实施例将更好地解释本发明。在任何情况下,以下实施例不应被认为是限制本发明的范围。
附图说明
图1.用于在大肠杆菌中表达K1K1的质粒。HGF/SF的K1结构域(aa 128-206)的两个拷贝以头-尾取向排列(串联)并在lac启动子的控制下表达。
图2.从大肠杆菌BL21培养物的包涵体中HisTrap纯化K1K1蛋白。(a)洗脱概览。在还原条件下通过SDS-PAGE分析对应于结合HisTrap的主蛋白峰(黑条)的级分(6、7、8、9和10),并显示在(b)中。
图3.K1K1蛋白的尺寸排阻层析。合并对应于来自His-Trap柱的主峰(图2)的级分,浓缩,并将等分试样装载在Superdex柱(a)上。在非还原(b)或还原条件(c)下,通过SDS-PAGE分析三个峰的级分。峰2和3主要含有K1K1蛋白(峰2含有二聚体的二聚体,而峰3含有预期的二聚体)。非还原性凝胶显示了从包涵体中洗涤前HisTrap纯化K1K1蛋白。
图4.K1K1蛋白的阳离子交换层析。合并对应于来自His-Trap柱的主峰(图2)的级分,浓缩,将等分试样装载在1ml Resource-S柱(a)上。在MDCK集落扩散测定中测试未结合材料(峰1)以及用NaCl梯度洗脱的级分的生物学活性。所有活性在峰2回收。
图5.K1K1刺激后的MET信号分析。用100和500pM HGF/SF(HGF),1、10和100nM K1K1和1、10和100nM NK1处理HeLa细胞。然后通过特异性总MET、Akt和ERK或磷酸化MET,磷酸化Akt和磷酸化ERK蛋白质印迹分析细胞裂解物。Ctrl:DMEM 0.1%FCS(胎牛血清)。
图6.MET-Fc-K1K1和ERK/Akt活化。(a)Alphascreen MET-Fc-K1K1。在384孔微量滴定板中进行K1K1与重组MET-Fc蛋白结合的交叉滴定测定。K1K1的终浓度为0-300nM,MET-Fc为0-10nM,链霉亲和素包被的供体珠和蛋白A缀合的受体珠为10μg/mL。(b)通过定量ALPHA测定的Akt和ERK磷酸化。细胞铺板,用不同的激动剂(HGF/SF,NK1,K1K1和K1B(生物素化的K1))刺激,然后在相同的96孔培养板中裂解。加入 UltraTM受体和供体珠并孵育2小时。使用Multimode PlateReader(PerkinElmer)上的标准Alpha设置测量发射信号强度。
图7.K1K1刺激后的MET信号分析。HeLa细胞用100pM HGF/SF,100nM K1K1或100nMNK1处理1、5、10或20分钟。然后通过特异性总MET,Akt和ERK或磷酸化MET,磷酸化Akt和磷酸化ERK蛋白质印迹分析细胞裂解物。
图8.K1K1蛋白的生物活性。测试K1K1蛋白(HisTrap汇集)的MDCK集落扩散活性,并与纯化的重组全长HGF/SF和NK1的MDCK集落扩散活性进行比较。数据分别为5(HGF/SF),7(K1K1)和7(NK1)个实验的平均值±标准偏差。
图9.K1K1蛋白诱导的细胞表型(细胞扩散测定)。将MDCK分离的细胞岛在100pMHGF/SF,100nM NK1、100nM K1K1的培养基中孵育。然后染色细胞,并在显微镜(100倍放大)下观察。
图10.K1K1蛋白诱导的细胞表型(MatrigelTM形态发生测定)。将MDCK细胞接种到MatrigelTM层上,并用100pM HGF/SF,100nM NK1和100nM K1K1处理。然后在培养一天后(a)和培养两天后(b),在40倍或100倍放大倍数下在显微镜下观察细胞。
图11.细胞表型(MTT测定)。MDCK细胞在有或没有茴香霉素(0.7μM)的培养基中和在500pM HGF/SF(HGF),100nM K1K1和100nM NK1的存在下培养过夜(15h)。然后进行MTT测定以评估细胞存活。结果表示为未处理对照的百分比。进行ANOVA检验以比较3种手段,P值<0.05认为具有统计学显著性。进行方差分析测试以比较所有手段,并且P值<0.001被认为表示统计学显著性差异。
图12.K1K1刺激后的MET信号分析。(A)用500pM HGF/SF(HGF),1nM K1K1,1nM变体1、1nM变体2、1nM变体3和1和100nM NK1处理HeLa细胞。(B)用10、100和500pM HGF/SF(HGF),100、500和1000pM变体1以及100、500和1000pM K1K1处理HeLa细胞。然后通过特异性总MET,Akt和ERK或磷酸化MET,磷酸化Akt和磷酸化ERK蛋白质印迹分析细胞裂解物。Ctrl:K1单体。
图13.通过定量ALPHA测定的Akt和ERK磷酸化。将细胞铺板,用不同激动剂(HGF/SF,NK1,K1K1和变体1、2和3)的递增浓度刺激10分钟,然后在相同的96孔培养板中裂解。加入反应混合物,并根据制造商方案(TGRES500和TGRA4S500)孵育2小时。使用Multimode Plate Reader(PerkinElmer)上的标准Alpha设置测量发射信号强度。
图14.K1K1蛋白诱导的细胞表型(细胞扩散测定)。MDCK分离的细胞岛在具有500pMHGF/SF,1、10和100nM K1K1、10nM K1K1变体1、2和3和100nM K1单体的培养基中培养。然后将细胞染色并在显微镜(100×)下观察。Ctrl:DMEM 10%FCS。
图15.K1K1蛋白诱导的细胞表型(MatrigelTM形态发生测定)。将MDCK细胞接种到MatrigelTM层上,并用1nM HGF/SF,100nM NK1、100nM K1K1、变体1、2或3和100nM K1单体处理。然后在显微镜(40x)下,在培养一(A)、二(B)和三(C)天后观察细胞。Ctrl:DMEM 10%FCS。
图16.K1K1蛋白诱导的细胞表型(MatrigelTM3D形态发生测定)。将MDCK细胞接种到1型胶原蛋白-MatrigelTM的层中,并将含有1nM HGF/SF(HGF),100nM K1K1、100nM K1K1变体1和100nM K1单体的培养基加入到该层上。将细胞固定,染色,然后在显微镜(100×)下观察。Ctrl:DMEM 10%FCS。
图17.K1K1变体1的体内MET活化(无标记)。小鼠注射(IV)5μg K1单体或5μg K1K1变体1。10分钟后,肝脏被提取,快速冷冻和粉碎。通过蛋白质印迹分析细胞裂解物中的MET,Akt和ERK磷酸化状态。
图18.K1K1和K1K1变体1的晶体结构和分子对齐(无标记)。K1K1变体1(无标记)的晶体结构,分辨率为已知负责结合MET受体的残基在图(A)中以点显示。K1K1(A)和K1K1变体1(无标记)的结构对齐(B)。计算的的RMSD值显示在图(B)的右下角。
图19.从大肠杆菌BL21培养物的包涵体中HisTrap纯化K1K1蛋白。在不同的纯化阶段细菌裂解液(大肠杆菌,BL21)和K1K1蛋白质的还原性SDS-PAGE。(A)泳道M:分子量标记;泳道1:超声处理后的不溶粗物质;泳道2:超声处理后的可溶性粗物质;泳道3:在2M L-精氨酸溶解72小时后的K1K1;泳道4:亲和层析纯化后的K1K1(HisTrap FF 5ml);泳道5:尺寸排阻层析后的K1K1。(B)从ncIBsinclusion体提取的K1K1(H6)蛋白的HisTrap层析。(C)从ncIBsinclusion体提取的K1K1(H6)蛋白的Superdex 75色谱。
图20.来自大肠杆菌BL21培养物的包涵体的K1K1变体1(无标记)的亲和层析纯化(HiTrapTM肝素HP柱)。将与主蛋白质峰对应的级分合并在一起,并浓缩至合适的体积用于制备型凝胶过滤层析。
图21.K1K1变体1(无标记)的尺寸排阻色谱。合并对应于来自HiTrapTM HeparinHP柱的主峰(图20)的级分,浓缩,并将等分试样装载到Superdex柱上。
实施例
实施例1.K1K1和K1K1变体的产生(无标记)
通过亚克隆含有HGF/SF的K1结构域(aa 128-206)的两个串联重复的DNA片段构建原核表达质粒(pET45b(+)-K1K1)(图1)。短接头连接第一和第二K1结构域。该构建体被命名为HGF/SF-K1K1(缩写为K1K1)。将原核表达质粒转染到BL21(DE3)细胞中。
成功转化后,开始蛋白质生产,在此阶段,细菌细胞在低浓度的IPTG(0.4mM)诱导后,于18℃生长24小时。在非常温和的提取程序之后,将含有K1K1(或其变体)的包涵体重新悬浮在含有高浓度L-精氨酸的缓冲液中,在4℃下孵育72小时以溶解并提取蛋白质。然后将提取的蛋白质通过亲和层析纯化,随后进行凝胶过滤层析步骤。
在25℃下用0.4mM IPTG过夜诱导BL21细胞之后,在富含非典型包涵体并用2M L-精氨酸溶解/复性的级分的HisTrap柱上的典型洗脱谱显示于图2。在~0.2M咪唑洗脱主峰(图2a),其主要含有K1K1蛋白(图2b)。
K1K1蛋白是非匀质的。Superdex 75上HisTrap汇集的尺寸排阻层析显示三个峰(图3a)。峰1表示容易解析并且不总是存在于HisTrap汇集中的少量高分子量污染物。峰2和3一直被观察到,并且都含有K1K1蛋白。峰3含有Superdex 75上预期洗脱体积的蛋白质和SDS-PAGE上预期的表观质量。峰2含有“二聚体的二聚体”,即两个K1K1分子,其可以与主K1K1峰分离,并且在非还原条件下在SDS-PAGE上较慢运行(图3b),但在还原性凝胶上不能与主K1K1蛋白区分开(图3c)。(补充结果如图19所示)。
通过阳离子交换层析证实了K1K1蛋白的HisTrap库的异质性(图4)。阳离子交换柱解析了三个峰,其中主峰(峰2)具有很强的生物活性。峰1和峰3在生物学上无活性。
缺少多组氨酸标签的其他K1K1变体通过使用肝素-琼脂糖亲和层析柱的亲和层析纯化。在使用L-精氨酸溶解后,K1K1变体1(无标记)的典型HisTrapTM肝素洗脱谱如图20所示。主峰含有高纯度的K1K1变体1(无标记),如凝胶过滤色谱图所示(图21)。
基于肝素亲和纯化随后凝胶过滤的方案与His-标签的存在无关。肝素允许纯化正确折叠的蛋白质,而镍柱的特异性/鉴别性较差。这个方案可以用于所有的变体。
实施例2.K1K1是强MET激动剂
K1K1的结合能力已通过使用重组MET-IgG1嵌合体的交叉滴定测定(图6A)确定,并且指示了K1K1和MET之间的结合。通过蛋白质印迹(图5)或ALPHAscreen定量方法(图6B)分析HGF/SF,K1K1,K1B(单体生物素化K1结构域)或重组NK1孵育后HeLa细胞中的MET活化和下游信号传导。通常,HGF/SF触发最大ERK和Akt活化至pM浓度。令人印象深刻的是,K1K1能够将ERK和Akt磷酸化水平触发到低nM范围,从而显示与NK1蛋白相似的激动剂活性。此外,K1K1在100nM诱导强烈的MET磷酸化。
还使用蛋白质印迹确定了MET和下游信号活化动力学(0-20分钟)(图7)。通常,HGF/SF在5和10分钟之间诱导出最大的MET自磷酸化,随后在约10-20分钟时产生最大的Akt和ERK磷酸化。相比之下,K1K1和NK1的MET磷酸化进行得更快,即在仅第一分钟内,然后降低。因此,仅在3-7分钟之后,观察到最大的ERK和Akt活化。
实施例3.K1K1促进细胞扩散,形态发生和存活表型
已经使用MDCK集落扩散测定法对K1K1蛋白的生物学活性进行了初步研究,并总结在图8中。来自7个不同表达实验的HisTrap库的活性已经与被广泛表征的天然全长HGF/SF和NK1(HGF/SF的片段)和进一步有用的基准进行比较。K1K1的活性比HGF/SF的活性在摩尔基准上低约3倍。
在HGF/SF(100pM)存在18-24h的情况下,MDCK细胞获得间质样表型并扩散。这种显著的表型也被NK1蛋白和K1K1诱导(图9)。
使用腔基底样基质(MatrigelTM)作为基底细胞外基质的模拟物进行进一步的细胞测定。在这些条件和无处理的情况下,MDCK细胞在24小时内自发形成MatrigelTM上的紧密球形团簇。相比之下,当用HGF/SF刺激时,MDCK细胞自组织成分支和连接的结构。值得注意的是,NK1和K1K1广泛促进了这种结构的形成(图10)。
研究了激动剂促进凋亡应激后细胞存活的能力。这种表型是HGF/SF的标志,其可以保护许多细胞类型免受血清消耗、紫外线辐射、局部缺血或一些化学物质引起的死亡。使用茴香霉素(一种诱导凋亡的DNA和蛋白质合成抑制剂)来胁迫MDCK细胞。16h后,茴香霉素处理诱导约90%的细胞死亡,而用HGF/SF预处理时,细胞死亡仅为~10%(图11)。用K1K1和NK1进行预处理导致约25%的细胞死亡,因此也可以显著地保护细胞。
实施例4.来自其他K1K1构建体的补充结果
通过蛋白质印迹(图12)和定量方法(图13)分析HGF/SF,K1K1(6xHis标签),K1K1变体1(无标记),K1K1变体2(长接头),K1K1变体3(GS接头)或NK1孵育时HeLa细胞中的MET活化和下游信号传导。通常,HGF/SF触发最大ERK和Akt活化至pM浓度。令人印象深刻的是,K1K1及其变体能够将ERK和Akt磷酸化水平触发到100pM范围,从而显示出比NK1强至少10倍的激动剂活性。此外,K1K1及其变体在100pM时开始诱导强烈的MET磷酸化。
在HGF/SF存在24小时的情况下,MDCK细胞获得间质样表型并扩散(图14)。这种显著的表型也被K1K1和变体1、2和3诱导。K1单体没有作用。
使用腔基底样基质(MatrigelTM)作为基底细胞外基质的模拟物进行进一步的细胞测定。在这些条件和无处理的情况下,MDCK细胞在24小时内自发形成MatrigelTM上的紧密球形团簇。相比之下,当用HGF/SF刺激时,MDCK细胞自组织成分支和连接的结构。值得注意的是,K1K1及其变体广泛促进了这种结构的形成(图15)。
类似地,当培养至胶原/MatrigelTM基质时,MDKC细胞自组织成分支结构。这些3D结构的侵入性和分支由HGF强烈增加。显然,K1K1和变体1促进了显著的3D形态发生(图16)。
最后,静脉注射K1单体或K1K1变体1,以观察其是否能活化肝脏(其是众所周知的强烈表达MET受体的器官)中的MET和下游通路。10分钟后,提取肝脏,通过蛋白质印迹法测定MET,ERK和Akt磷酸化状态(图17)。K1K1变体1注射诱导与肝脏中强烈的Akt和ERK活化相关的清晰的MET磷酸化。相反,K1对照导致无法检测到信号。
实施例5.K1K1变体1(无标记)的3D结构
K1K1和K1K1变体1(无标签形式)的晶体结构分别通过X射线晶体学解析,分辨率分别为1.4和1.8这些结构显示了一个非常有趣和重要的事实:在这两种情况下,K1K1分子都采用扩展构象,在外部和相对侧暴露出两个MET结合位点(图18中的图A)。这证实了产生能够以形成活性信号传导复合物的正确取向结合两个受体的分子的效用。从K1K1和K1K1变体1的3D结构的对齐,它们看起来几乎相同,其中RMSD(原子位置的均方根差)低于一个AV(图18的图B)。
方法
载体构建
通过在头尾(N-ter至C-ter)取向上亚克隆含有两个拷贝的K1结构域的DNA片段来构建原核表达质粒(pET45b(+)-K1K1)。从产生用于在酵母巴斯德毕赤酵母(P.pastoris)中表达K1K1的另一表达质粒(pPIC9K-K1K1)通过PCR扩增编码K1K1的cDNA序列。为了组装表达载体(pET45b(+)-K1K1),先前通过两个人HGF/SF kringle 1结构域的融合产生了K1K1cDNA,该结构域各自先前通过PCR技术扩增。为了扩增K1K1的N末端单体,使用以下引物组:正向引物P1(5'-ATCATCCCATGGCCATTAGAAACTGCATCATTGGTAAAGGACG-3')(SEQ ID NO:22)和反向引物P3(5'-TTCAACTTCTGAACACTGAGGA-3')(SEQ ID NO:20)。对于C末端单体,使用下列引物对:正向引物P4(5'-CAGAAGTTGAATGCATCATTGGTGAAGGA-3')(SEQ ID NO:21)和反向引物P2(5'-ACAGCGGCCGCTCATCAA-3')(SEQ ID NO:23)。为了使N末端和C末端K1cDNA融合,正向引物P3和反向引物P4均携带Hpy188I限制性位点。
在下列引物对中,正向引物P1(5'-ATCATCCCATGGCCATTAGAAACTGCATCATTGGTAAAGGACG-3')(SEQ ID NO:22)和反向引物P2(5'-ACAGCGGCCGCTCATCAA-3')(SEQ ID NO:23)分别携带NcoI和NotI位点,以便将K1K1cDNA***到表达载体中。PCR条件由94℃15秒,54℃30秒和68℃60秒的28个循环组成,用于DNA扩增的酶是Pfx DNA聚合酶(Invitrogen)。将PCR扩增的DNA片段在含有溴化乙锭(EB)的1.5%琼脂糖凝胶上分离,并在(长波长)UV光照射下显现。使用ZymocleanTM凝胶DNA回收试剂盒(Zymogen)从琼脂糖凝胶回收并纯化该条带,并使用NcoI(New England Biolabs)和NotI(New England Biolabs)消化。用NcoI和NotI限制性消化pET45b(+)质粒,去磷酸化并从1%琼脂糖凝胶中分离。使用DNA Clean&ConcentratorTM试剂盒(Zymogen)从琼脂糖凝胶回收和纯化消化产物,随后使用快速连接酶试剂盒(New England Biolabs)将K1K1cDNA***打开的载体中。将连接产物在大肠杆菌MACH1细胞(New England Biolabs)中转化,并将细菌在含有氨苄青霉素的LB琼脂平板上生长过夜。使用T7通用正向引物(5'-TAATACGACTCACTATAGGG-3')(SEQ ID NO:24)退火和引物P2(SEQ ID NO:23)作为反向引物,通过PCR筛选单个菌落。通过在两条链上整个构建体的测序来确认K1K1构建体中的kringle结构域的正确取向和无人工突变的确认。
大肠杆菌表达
K1K1蛋白及其变体含有总共6个二硫键(每个kringle结构域3个)。在大肠杆菌中生产富含二硫键的蛋白质存在显著的挑战,并且在大多数情况下,这样的蛋白质聚集在称为包涵体的大聚集物中。典型的包涵体具有0.5-1.3μm的直径,并且发现在细菌细胞质内或周质中。包涵体主要由靶蛋白组成(从50到90%),尽管其他细胞质蛋白和其他细胞成分几乎总是与它们相关联的。然而,包涵体还可以含有正确折叠的靶蛋白,特别是当培养物在低温下生长时。这些“非典型”包涵体富含靶蛋白的正确折叠前体,其可在非变性条件下有效提取。
为了表达正确折叠和生物活性的K1K1,本文采用后一种策略。简言之,质粒pET45b(+)-K1K1用于转化大肠杆菌DH5α菌株(用于繁殖)和BL21菌株(用于表达)。BL21细胞在含有氨苄青霉素的LB培养基中于37℃培养直到细胞达到0.5至0.6之间的OD 600,此时用0.4mM异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)诱导表达。诱导后,细胞在摇动的培养箱(250rpm)中于18℃生长24小时。通过在4℃下以5000g离心30分钟收集细胞,重悬于PBS中并通过超声处理裂解(以40秒间隔的20秒10次循环)。或者,使用溶菌酶在37℃裂解细胞1小时,然后进行几次冷冻和解冻循环或使用Emulsyflex机械裂解。超声处理或溶菌酶处理后,细胞裂解物在4℃以5000g离心30分钟,重新悬浮于含有0.4%(v/v)Triton X-100的PBS中,并在室温下温育1小时伴以温和搅拌以除去非特异性吸附的蛋白质。将裂解物在4℃下以5000g再次离心30分钟,将沉淀物重新悬浮于含有0.025%(v/v)苯氧基聚乙氧基乙醇(NP40)的PBS中,并在4℃下在温和搅拌下孵育1小时,以除去额外的非特异性吸附的蛋白质,然后进行新的离心。最后,将沉淀物在冰冷的PBS中洗涤数次,以除去痕量的去垢剂。为了用L-精氨酸溶解和复性K1K1蛋白质,将获得的由几乎纯的包涵体组成的沉淀物重新悬浮于含有2M L-精氨酸的PBS中,并在37℃下以250rpm摇动孵育过夜。
亲和层析
用2M L-精氨酸过夜孵育和进一步的离心步骤(在4℃下5000g,30分钟)后得到的上清液通过0.22μm过滤器过滤,并以2ml/min装载到用调节至500mM NaCl的PBS平衡的1mlHisTrap粗FF亲和柱上。洗涤柱直到基线回到零,并用调节至500mM NaCl的PBS中的130ml咪唑梯度(0-500mM)洗脱结合的蛋白质。通过SDS-PAGE、在Superdex 75 10/30(GEHealthcare)柱上的尺寸排阻色谱分析含蛋白质的级分,并使用MDCK集落扩散测定法进行生物学活性的分析(Stoker,M.et al.,Scatter factor is a fibroblast-derivedmodulator of epithelial cell mobility.Nature 327,239-242(1987))。
或者,将用2M L-精氨酸孵育72小时和进一步的离心步骤(在4℃下15000g,30分钟)后获得的上清液稀释100倍到装载缓冲液(25mM Tris pH7.4、500mM NaCl)中并通过0.22μm的过滤器过滤。以这种方式制备的样品以1.5ml/min的流速加载到柱(5mlHisTrapTM-FF)上过夜。将柱洗涤直到记录的UV迹线显示平坦的低吸光度基线。然后用10个柱体积的50%缓冲液B(25mM Tris,pH7.4、500mM NaCl1M咪唑)以步骤梯度洗脱以5ml/min洗脱结合的物质,然后在一个步骤中达到100%缓冲液B并保持另外10个柱体积。在280nm处监测UV吸收,并在整个洗脱程序中收集5毫升的级分。
凝胶过滤层析
HiLoadTM 16/60 SuperdexTM 75用柱缓冲液(25mM Tris,500mM NaCl,pH 7.4)平衡。将来自亲和层析步骤的浓缩级分用5ml环加载到柱上。以0.5ml/min的流速进行色谱分析,并在整个洗脱程序中收集5ml级分。收集对应于预期峰的级分,合并在一起,分析,并且如果需要,将其冷冻储存在-80℃。
阳离子交换层析
将来自包涵体的可溶性变体1蛋白和含有来自His-Trap柱的级分(K1K1,变体2和变体3)的蛋白质合并,并在4℃下针对50mM MES,150mM NaCl pH 6.0透析24小时。将样品离心,通过0.22μm过滤器过滤并以0.5ml/min的速度加载到在50mM MES,150mM NaCl pH6.0中平衡的1ml Resource-S柱(GE Healthcare)上。结合的蛋白质用NaCl的梯度(0.25-100M)洗脱。通过SDS-PAGE和MDCK集落扩散测定法收集和分析级分。
MET信号通路和剂量反应(蛋白质印迹)
用500pM HGF,1nM K1K1、1nM K1K1变体1、2和3或1或100nM NK1处理HeLa细胞10分钟。然后通过特异性总MET,Akt和ERK或磷酸化MET,磷酸化Akt和磷酸化ERK蛋白质印迹分析细胞裂解物。通过刮擦收集细胞,然后用补充有新鲜添加的蛋白酶和磷酸酶抑制剂(Sigma)的裂解缓冲液(20mM HEPES pH 7.4、142mM KCl,5mM MgCl2,1mM EDTA,5%甘油,1%NP40和0.1%SDS)在冰上裂解。通过离心(20000g×15min)澄清裂解液,测定蛋白质浓度(BCA蛋白测定试剂盒,Pierce,Thermo Scientific,IL,USA)。通过经典的SDS-PAGE或NuPAGE(4-12%或10%Bis-Tris预制凝胶)(Life technologies)分离相同蛋白质量的细胞提取物并电转移到聚偏二氟乙烯(PVDF)膜(Merck Millipore)上。用指定的一抗检测膜,随后用合适的HRP缀合的二抗孵育。使用West Dura Extended Duration Substrate X射线胶片(CL-XposureTM Film,Thermo Scientific),通过化学发光观察蛋白质-抗体复合物。
对于剂量反应,用10、100和500pM HGF/SF(HGF),100、500和1000pM K1K1变体1和100、500和1000pM K1K1处理HeLa细胞10分钟。
动力学(蛋白质印迹)
HeLa细胞用100pM HGF,100nM K1K1和100nM NK1处理1、5、10或20分钟。然后通过特异性总MET,Akt和ERK或磷酸化MET,磷酸化Akt和磷酸化ERK蛋白质印迹分析细胞裂解物。通过刮擦收集细胞,然后用补充有新鲜添加的蛋白酶和磷酸酶抑制剂(Sigma)的裂解缓冲液(20mM HEPES pH 7.4、142mM KCl,5mM MgCl2,1mM EDTA,5%甘油,1%NP40和0.1%SDS)在冰上裂解。通过离心(20000g×15min)澄清裂解液,测定蛋白质浓度(BCA蛋白测定试剂盒,Pierce,Thermo Scientific,IL,USA)。通过经典的SDS-PAGE或NuPAGE(4-12%或10%Bis-Tris预制凝胶)(Life technologies)分离相同蛋白质量的细胞提取物并电转移聚偏二氟乙烯(PVDF)膜(Merck Millipore)上。用指定的一抗检测膜,随后用合适的HRP缀合的二抗孵育。使用X射线胶片(CL-XposureTM Film,Thermo Scientific),使用West Dura Extended Duration Substrate(Thermo Scientific)通过化学发光显示蛋白质-抗体复合物。
MDCK扩散
将MDCK分离的细胞岛在具有100pM HGF/SF,100nM K1K1和100nM NK1的培养基中孵育18-24小时。或者,将MDCK分离的细胞岛在具有500pM HGF/SF,10nM K1K1和变体1、2或3和100nM K1单体的培养基中孵育24小时。然后染色细胞并在显微镜(100×)下观察。
将细胞以低密度接种(在12孔板中2000个细胞/孔)以形成紧密的菌落。处理后,当观察到菌落扩散时,根据制造商的说明,将细胞固定并用染色剂(Merck,Darmstadt,Germany)着色。使用100倍放大倍数的相差显微镜(Nikon Eclipse TS100,Tokyo,Japan)捕获代表性的图像。
MDCK形态发生
将MDCK细胞接种到Growth Factor Reduced MatrigelTM(BD Biosciences)(24孔板的100000个细胞/孔)上,用100pM HGF/SF,100nM K1K1和100nM NK1处理18-24小时,并在相差显微镜下观察。用40倍和100倍放大倍率捕获代表性的图像(Nikon Eclipse TS100)。
或者,将MDCK细胞接种到15孔显微载玻片血管生成中的Growth FactorReduced MatrigelTM(BD Biosciences)的10μL层上(2500个细胞/孔),并用50μL的1nM HGF/SF,100nM K1K1变体1、2或3和100nM K1单体处理。在相差显微镜下在24、48和72h(40x)观察细胞。代表性的图像以40倍放大倍率(Nikon Eclipse TS100)拍摄。
MDCK 3D形态发生
将MDCK细胞接种在由含有1nM HGF/SF(HGF)、100nM NK1、100nM K1K1、变体1和100nM K1单体的培养基覆盖的24孔板中的1型胶原蛋白-MatrigelTM的厚层中。将细胞固定,用伊文思蓝(0.01%)染色,然后在相差(Nikon Eclipse TS100)和共聚焦(Leica LSM 880)显微镜(Ex405nm/Em630)下观察。代表性的图像以40倍或100倍的倍率捕获,并且在Z-叠层中3D重构。
MDCK存活
将MDCK细胞在含有或不含茴香霉素(0.7μM)的0.1%FBS的培养基中并在500pMHGF/SF、100nM K1K1和100nM NK1存在下培养过夜(15h)。然后进行MTT测定以评估细胞存活。结果表示为未处理对照的百分比。
用PBS洗涤细胞以除去死细胞,然后在含有0.5mg/ml 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT,Invitrogen)的培养基中孵育1小时。在用PBS洗涤的步骤后,将甲臜晶体溶解并与0.04M HCl在异丙醇中充分混合。对于每个条件,将60μl甲臜溶液一式三份加载到96孔板上。然后用微板分光光度计分别在550nm和620nm(作为测试和参考波长)测量吸光度。吸光度与细胞数相关。
交叉滴定法
在384孔微量滴定板(OptiPlateTM-384,CA,USA,50μL最终反应体积)中进行K1K1与重组MET-Fc蛋白结合的交叉滴定测定。K1K1的终浓度为0-300nM,MET-Fc为0-10nM,链霉亲和素包被的供体珠和蛋白A缀合的受体珠为10μg/mL。用于制备所有蛋白质溶液和珠悬浮液的缓冲液是:PBS,5mM HEPES pH7.4,0.1%BSA。将板在暗箱中在23℃下孵育60分钟。使用Multimode Plate Reader(PerkinElmer)上的标准Alpha设置测量发射信号强度。
通过
UltraTM方法进行Akt和ERK磷酸化测定
根据UltraTM(CA,USA)中提及的制造商方案进行测定。简言之,将细胞接种,用不同的激动剂(HGF/SF,NK1,K1K1和K1B(生物素化的K1))刺激7分钟,然后在相同的96孔培养板中裂解。将溶解物(10μL)转移到384孔微量培养板中,用于检测磷酸化Akt(ALSU-PAKT-B500,Ser473)和ERK(ALSU-PERK-A500,Thr202/Tyr204)。加入UltraTM受体和供体珠并孵育2小时。使用Multimode Plate Reader(PerkinElmer)上的标准Alpha设置测量发射信号强度。
通过ALPHAScreen
方法进行Akt和ERK磷酸化测定
根据ERK(TGRES500)和Akt(TGRA4S500)(CA,USA)中提及的制造商方案进行测定。简言之,将细胞铺板,用不同的激动剂(HGF/SF,NK1,K1K1,变体1、2或3和K1(生物素化的K1))刺激10分钟,然后在相同的96孔培养板中裂解。将裂解物(5μL)转移到384孔微量培养板中,用于检测磷酸化Akt(Ser473)和ERK(Thr202/Tyr204)。加入受体和供体珠混合物,并根据制造商的程序孵育2小时。使用Multimode Plate Reader(PerkinElmer)上的标准Alpha设置测量发射信号强度。
体内MET信号传导活化:
为了可视化肝脏中的MET活化,使用重量为19-21g的FVB小鼠(n=2)(CharlesRiver)。用异氟烷(Aerrane,Baxter,USA)麻醉后,静脉内注射在PBS中5μg的K1单体或K1K1变体1。10分钟后处死小鼠,用补充有蛋白酶和磷酸酶抑制剂的PBS灌注肝脏。提取肝脏并通过蛋白质印迹分析MET、ERK和Akt活化。
Fas诱发暴发性肝炎
使用体重19-21g的FVB小鼠进行实验。用异氟烷麻醉后,给小鼠静脉注射125ng/g体重的与不同的激动剂如K1K1变体1(无标记)、HGF/SF、NK1混合的抗Fas抗体(Clone Jo-2,CD95,Pharmingen,BD Biosciences)。在第一次注射后90分钟,用每种激动剂第二次注射小鼠。另外3小时后处死小鼠,并用补充有蛋白酶和磷酸酶抑制剂的PBS灌注它们的肝脏。
同时,为了可视化肝脏中的MET活化,给小鼠静脉注射每种激动剂10分钟。
对于组织学分析,收集肝组织,在4%多聚甲醛中固定过夜,并在异戊烷中快速冷冻,浸没在液氮中,并嵌入OCT(Tissue-VWR,PA,USA)。冷冻肝切片(5μm)用苏木精和伊红(HE)染色用于一般形态学观察。还根据制造商的说明书(Apoptag FluoresceinDirect In Situ试剂盒,Merck Millipore,Billerica,MA,USA)在肝切片上进行TUNEL染色以测定细胞凋亡。对于分子分析,将提取的肝组织立即在液氮中冷冻。肝脏在补充有新鲜添加的蛋白酶和磷酸酶抑制剂的裂解缓冲液中破碎。
体内“伤口愈合”实验方案
动物模型是猪(3只猪用于研究)。
膳宿包括7天的适应环境+25天的随访。
临床观察和称重在4周内每周进行一次。
伤口愈合模型:
-在动物的两侧制造方形的皮肤-表皮创伤(2x2cm)。
-每只动物是自己的见证
ο第一侧=用制剂处理
ο第二侧=阴性对照,用安慰剂治疗
-处理:两种浓度(1和50nM)的K1K1 TL(无标记)的2种制剂
ο在4周内每周两次
临床观察和称重:
研究以下参数后进行伤口愈合评估:
-伤口闭合-形态测量研究(伤口图片)
-组织活检(HES和MTG)-血管生成-肉芽组织
-D7、D14和D35的上皮形成
-安乐死。
序列表
<110> Universit?Sciences et Technologies de Lille - Lille I
Centre National de la Recherche Scientifique
Universit?de Lille 2 Droit et Sant?
Institut Pasteur de Lille
Universita Degli Studi Di Pavia
<120> MET受体激动蛋白
<130> WOB 14 CA LIL K1K1
<150> EP 15152027.7
<151> 2015-01-21
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 79
<212> PRT
<213> 智人
<400> 1
Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr
1 5 10 15
Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu
20 25 30
His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn
35 40 45
Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr
50 55 60
Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys
65 70 75
<210> 2
<211> 74
<212> PRT
<213> 智人
<400> 2
Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr
1 5 10 15
Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu
20 25 30
His Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro
35 40 45
Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val
50 55 60
Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys
65 70
<210> 3
<211> 238
<212> DNA
<213> 智人
<400> 3
tgcatcattg gtaaaggacg cagctacaag ggaacagtat ctatcactaa gagtggcatc 60
aaatgtcagc cctggagttc catgatacca cacgaacaca gctttttgcc ttcgagctat 120
cggggtaaag acctacagga aaactactgt cgaaatcctc gaggggaaga agggggaccc 180
tggtgtttca caagcaatcc agaggtacgc tacgaggtct gtgacattcc tcagtgtt 238
<210> 4
<211> 223
<212> DNA
<213> 智人
<400> 4
tgcatcattg gtaaaggacg cagctacaag ggaacagtat ctatcactaa gagtggcatc 60
aaatgtcagc cctggagttc catgatacca cacgaacaca gctatcgggg taaagaccta 120
caggaaaact actgtcgaaa tcctcgaggg gaagaagggg gaccctggtg tttcacaagc 180
aatccagagg tacgctacga ggtctgtgac attcctcagt gtt 223
<210> 5
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 接头的肽序列
<400> 5
Ser Glu Val Glu
1
<210> 6
<211> 12
<212> DNA
<213> 人工序列
<220>
<223> 接头的核苷酸序列
<400> 6
tcagaagttg aa 12
<210> 7
<211> 162
<212> PRT
<213> 人工序列
<220>
<223> K1K1
<400> 7
Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr
1 5 10 15
Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu
20 25 30
His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn
35 40 45
Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr
50 55 60
Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser
65 70 75 80
Glu Val Glu Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val
85 90 95
Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile
100 105 110
Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu
115 120 125
Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp
130 135 140
Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro
145 150 155 160
Gln Cys
<210> 8
<211> 152
<212> PRT
<213> 人工序列
<220>
<223> K1K1
<400> 8
Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr
1 5 10 15
Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu
20 25 30
His Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro
35 40 45
Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val
50 55 60
Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Ile
65 70 75 80
Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser
85 90 95
Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His Ser
100 105 110
Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly
115 120 125
Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr
130 135 140
Glu Val Cys Asp Ile Pro Gln Cys
145 150
<210> 9
<211> 177
<212> PRT
<213> 人工序列
<220>
<223> K1K1
<400> 9
Met Ala Ile Arg Asn Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly
1 5 10 15
Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser
20 25 30
Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys
35 40 45
Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly
50 55 60
Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp
65 70 75 80
Ile Pro Gln Cys Ser Glu Val Glu Cys Ile Ile Gly Lys Gly Arg Ser
85 90 95
Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro
100 105 110
Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr
115 120 125
Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu
130 135 140
Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu
145 150 155 160
Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu His His His His His
165 170 175
His
<210> 10
<211> 534
<212> DNA
<213> 人工序列
<220>
<223> 编码K1K1的核苷酸序列
<400> 10
atggccatta gaaactgcat cattggtaaa ggacgcagct acaagggaac agtatctatc 60
actaagagtg gcatcaaatg tcagccctgg agttccatga taccacacga acacagcttt 120
ttgccttcga gctatcgggg taaagaccta caggaaaact actgtcgaaa tcctcgaggg 180
gaagaagggg gaccctggtg tttcacaagc aatccagagg tacgctacga ggtctgtgac 240
attcctcagt gttcagaagt tgaatgcatc attggtaaag gacgcagcta caagggaaca 300
gtatctatca ctaagagtgg catcaaatgt cagccctgga gttccatgat accacacgaa 360
cacagctttt tgccttcgag ctatcggggt aaagacctac aggaaaacta ctgtcgaaat 420
cctcgagggg aagaaggggg accctggtgt ttcacaagca atccagaggt acgctacgag 480
gtctgtgaca ttcctcagtg tagtgaagtt gaacatcatc atcatcatca ttga 534
<210> 11
<211> 5683
<212> DNA
<213> 人工序列
<220>
<223> 编码K1K1的质粒
<400> 11
ggccgcactc gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga 60
ctcgtctact agcgcagctt aattaaccta ggctgctgcc accgctgagc aataactagc 120
ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 180
atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 240
gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 300
ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 360
cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 420
gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 480
tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 540
gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 600
ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttctggc 660
ggcacgatgg catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 720
gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 780
taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 840
tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 900
tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 960
gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 1020
gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 1080
ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 1140
cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 1200
tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 1260
cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 1320
agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 1380
cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 1440
aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 1500
aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 1560
gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 1620
gaatactcat actcttcctt tttcaatcat gattgaagca tttatcaggg ttattgtctc 1680
atgagcggat acatatttga atgtatttag aaaaataaac aaataggtca tgaccaaaat 1740
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1800
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1860
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 1920
cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 1980
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 2040
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 2100
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 2160
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 2220
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 2280
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 2340
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 2400
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 2460
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 2520
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 2580
gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat atggtgcact 2640
ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac 2700
gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 2760
cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 2820
gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag 2880
cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt 2940
tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt 3000
cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac 3060
cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac 3120
tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca 3180
ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc 3240
agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca 3300
gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt 3360
tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa 3420
ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc 3480
cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc 3540
ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc 3600
agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 3660
gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga 3720
ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc 3780
agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg 3840
gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg 3900
gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg 3960
ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc 4020
cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc 4080
agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat 4140
gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg 4200
atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc 4260
acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc 4320
gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac 4380
accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac 4440
ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc 4500
agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt 4560
tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa 4620
gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg 4680
aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg 4740
gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag 4800
taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc 4860
caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag 4920
cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac 4980
cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga 5040
tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc 5100
ccctctagaa ataattttgt ttaactttaa gaaggagata taccatggcc attagaaact 5160
gcatcattgg taaaggacgc agctacaagg gaacagtatc tatcactaag agtggcatca 5220
aatgtcagcc ctggagttcc atgataccac acgaacacag ctttttgcct tcgagctatc 5280
ggggtaaaga cctacaggaa aactactgtc gaaatcctcg aggggaagaa gggggaccct 5340
ggtgtttcac aagcaatcca gaggtacgct acgaggtctg tgacattcct cagtgttcag 5400
aagttgaatg catcattggt aaaggacgca gctacaaggg aacagtatct atcactaaga 5460
gtggcatcaa atgtcagccc tggagttcca tgataccaca cgaacacagc tttttgcctt 5520
cgagctatcg gggtaaagac ctacaggaaa actactgtcg aaatcctcga ggggaagaag 5580
ggggaccctg gtgtttcaca agcaatccag aggtacgcta cgaggtctgt gacattcctc 5640
agtgtagtga agttgaacat catcatcatc atcattgatg agc 5683
<210> 12
<211> 728
<212> PRT
<213> 智人
<400> 12
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu
1 5 10 15
Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln
20 25 30
Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45
Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60
Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu
65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys
85 90 95
Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe
100 105 110
Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys
115 120 125
Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140
Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His
145 150 155 160
Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr
165 170 175
Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser
180 185 190
Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu
195 200 205
Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp
210 215 220
His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro
225 230 235 240
His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp
245 250 255
Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr
260 265 270
Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys
275 280 285
Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu
290 295 300
Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile
305 310 315 320
Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu
325 330 335
His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn
340 345 350
Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr
355 360 365
Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp
370 375 380
Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met
385 390 395 400
Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp
405 410 415
Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala
420 425 430
Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His
435 440 445
Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys
450 455 460
Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu
465 470 475 480
Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val
485 490 495
Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg
500 505 510
Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp
515 520 525
Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr
530 535 540
Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys
545 550 555 560
Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly
565 570 575
Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp
580 585 590
Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu
595 600 605
Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn
610 615 620
Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu
625 630 635 640
Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu
645 650 655
Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp
660 665 670
Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu
675 680 685
Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly
690 695 700
Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile
705 710 715 720
Leu Thr Tyr Lys Val Pro Gln Ser
725
<210> 13
<211> 171
<212> PRT
<213> 人工序列
<220>
<223> K1K1变体1
<400> 13
Met Ala Ile Arg Asn Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly
1 5 10 15
Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser
20 25 30
Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys
35 40 45
Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly
50 55 60
Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp
65 70 75 80
Ile Pro Gln Cys Ser Glu Val Glu Cys Ile Ile Gly Lys Gly Arg Ser
85 90 95
Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro
100 105 110
Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr
115 120 125
Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu
130 135 140
Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu
145 150 155 160
Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu
165 170
<210> 14
<211> 183
<212> PRT
<213> 人工序列
<220>
<223> K1K1变体2
<400> 14
Met Ala Ile Arg Asn Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly
1 5 10 15
Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser
20 25 30
Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys
35 40 45
Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly
50 55 60
Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp
65 70 75 80
Ile Pro Gln Cys Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser Gly
85 90 95
Gly Gly Gly Ser Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr
100 105 110
Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met
115 120 125
Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp
130 135 140
Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro
145 150 155 160
Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile
165 170 175
Pro Gln Cys Ser Glu Val Glu
180
<210> 15
<211> 171
<212> PRT
<213> 人工序列
<220>
<223> K1K1变体3
<400> 15
Met Ala Ile Arg Asn Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly
1 5 10 15
Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser
20 25 30
Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys
35 40 45
Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly
50 55 60
Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp
65 70 75 80
Ile Pro Gln Cys Gly Ser Gly Gly Cys Ile Ile Gly Lys Gly Arg Ser
85 90 95
Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro
100 105 110
Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr
115 120 125
Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu
130 135 140
Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu
145 150 155 160
Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu
165 170
<210> 16
<211> 177
<212> PRT
<213> 人工序列
<220>
<223> K1K1变体4
<400> 16
Met Ala Ile Arg Asn Cys Ile Ile Gly Glu Gly Glu Ser Tyr Lys Gly
1 5 10 15
Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser
20 25 30
Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys
35 40 45
Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly
50 55 60
Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp
65 70 75 80
Ile Pro Gln Cys Ser Glu Val Glu Cys Ile Ile Gly Glu Gly Glu Ser
85 90 95
Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys Cys Gln Pro
100 105 110
Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro Ser Ser Tyr
115 120 125
Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg Gly Glu
130 135 140
Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg Tyr Glu
145 150 155 160
Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu His His His His His
165 170 175
His
<210> 17
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 17
Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 18
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 切割位点
<400> 18
Leu Val Pro Arg Gly Ser
1 5
<210> 19
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 19
Gly Ser Gly Gly Ser
1 5
<210> 20
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 20
ttcaacttct gaacactgag ga 22
<210> 21
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 21
cagaagttga atgcatcatt ggtgaagga 29
<210> 22
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 22
atcatcccat ggccattaga aactgcatca ttggtaaagg acg 43
<210> 23
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 23
acagcggccg ctcatcaa 18
<210> 24
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 24
taatacgact cactataggg 20
Claims (15)
1.含有两个肽结构域的蛋白质,所述肽结构域分别命名为K1a和K1b,所述肽结构域K1a和K1b各自包含肝细胞生长因子/扩散因子的K1肽结构域,
所述K1肽结构域由与SEQ ID NO:1具有至少80%同一性,优选至少90%同一性的序列组成,
所述蛋白质能够诱导酪氨酸激酶受体MET的活化,
条件是所述蛋白质不包含HGF/SF的N末端结构域。
2.根据权利要求1所述的蛋白质,其中所述肽结构域K1a和K1b是相同的。
3.根据权利要求1或2中任一项所述的蛋白质,其中所述肽结构域K1a和K1b各自由选自氨基酸序列SEQ ID NO:1和SEQ ID NO:2的氨基酸序列组成。
4.根据权利要求1至3中任一项所述的蛋白质,所述蛋白质包含连接K1a和K1b的肽接头,所述肽接头优选由1至50个氨基酸,更优选10至20个氨基酸构成。
5.根据权利要求1至4中任一项所述的蛋白质,所述蛋白质包含或由下列组成:氨基酸序列SEQ ID NO:7或与SEQ ID NO:7具有至少80%,优选90%同一性的氨基酸序列。
6.编码权利要求1至5中任一项所定义的蛋白质的核酸分子,所述核酸优选由核酸序列SEQ ID NO:10组成。
7.含有如权利要求6所定义的核酸分子的表达载体,所述载体优选包含核酸序列SEQID NO:11或由核酸序列SEQ ID NO:11组成。
8.含有如权利要求7所述的表达载体的宿主细胞,所述宿主细胞优选选自酵母细胞和细菌细胞。
9.组合物,其包含:
权利要求1至5中任一项所定义的蛋白质,或
-编码所述蛋白质的核酸分子,或
-含有所述核酸分子的表达载体,或
-含有所述表达载体的宿主细胞。
10.如权利要求1至5中任一项所述的蛋白质,其用于诊断方法。
11.如权利要求1至5中任一项所述的蛋白质,其用于医学成像。
12.如权利要求1至5中任一项所定义的蛋白质,其用作药物。
13.根据权利要求1至5中任一项所述的蛋白质,其用于通过促进细胞存活或组织再生来治疗组织损伤。
14.根据权利要求1至5中任一项所述的蛋白质,其用于治疗选自以下的病状:上皮器官疾病,包括急性和慢性肝脏疾病、急性和慢性肾脏疾病、慢性肺部疾病和慢性皮肤伤口,中枢神经***疾病,包括神经元疾病和硬化,缺血性心脏病,外周血管疾病,糖尿病和相关并发症如周围神经病变。
15.获得如权利要求1至5中任一项所述的包含至少两个K1肽结构域的蛋白质的方法,包括以下步骤:
-在表达载体中***编码包含至少两个K1肽结构域,优选两个K1肽结构域的重组蛋白的核酸序列,
-将所述载体克隆在宿主细胞中,并表达所述重组蛋白,
-提取和纯化所述重组蛋白,所述重组蛋白是包含至少两个K1结构域的蛋白质。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP15152027 | 2015-01-21 | ||
EP15152027.7 | 2015-01-21 | ||
PCT/EP2016/051267 WO2016116577A1 (en) | 2015-01-21 | 2016-01-21 | Met receptor agonist proteins |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107531770A true CN107531770A (zh) | 2018-01-02 |
CN107531770B CN107531770B (zh) | 2021-10-08 |
Family
ID=52434559
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201680006622.2A Active CN107531770B (zh) | 2015-01-21 | 2016-01-21 | Met受体激动蛋白 |
Country Status (11)
Country | Link |
---|---|
US (1) | US10435450B2 (zh) |
EP (1) | EP3247719B1 (zh) |
JP (1) | JP6944875B2 (zh) |
CN (1) | CN107531770B (zh) |
CA (1) | CA2973300C (zh) |
DK (1) | DK3247719T3 (zh) |
ES (1) | ES2761852T3 (zh) |
HU (1) | HUE047148T2 (zh) |
PL (1) | PL3247719T3 (zh) |
PT (1) | PT3247719T (zh) |
WO (1) | WO2016116577A1 (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002088354A1 (en) * | 2001-04-27 | 2002-11-07 | Medical Research Council | The nk1 fragment of hepatocyte growth factor/scatter factor (hgf/sf) and variants thereof, and their use |
US20060003931A1 (en) * | 2004-05-06 | 2006-01-05 | Genentech, Inc. | Crystal structure of the hepatocyte growth factor and methods of use |
CN101356189A (zh) * | 2005-11-10 | 2009-01-28 | 受体生物公司 | 肝细胞生长因子内含子融合蛋白 |
US20090215686A1 (en) * | 2007-03-05 | 2009-08-27 | Huaqiang Eric Xu | Nk1-based polypeptides and related methods |
WO2011116396A2 (en) * | 2010-03-19 | 2011-09-22 | The Board Of Trustees Of The Leland Stanford Junior University | Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090202606A1 (en) | 2008-01-25 | 2009-08-13 | Viromed Co., Ltd. | Treatment and Prevention of Cardiac Conditions Using Two or More Isoforms of Hepatocyte Growth Factor |
WO2009125986A2 (en) | 2008-04-09 | 2009-10-15 | Viromed Co., Ltd. | Lyophilized dna formulations for enhanced expression of plasmid dna |
WO2011103382A2 (en) * | 2010-02-22 | 2011-08-25 | The Brigham And Women's Hospital | Compositions and methods for inducing angiogenesis |
MX2014005318A (es) | 2011-11-03 | 2015-01-14 | Viromed Co Ltd | Terapia genica para la neuropatia diabetica usando una isoforma hgf. |
-
2016
- 2016-01-21 ES ES16701312T patent/ES2761852T3/es active Active
- 2016-01-21 CN CN201680006622.2A patent/CN107531770B/zh active Active
- 2016-01-21 HU HUE16701312A patent/HUE047148T2/hu unknown
- 2016-01-21 JP JP2017539030A patent/JP6944875B2/ja active Active
- 2016-01-21 PT PT167013127T patent/PT3247719T/pt unknown
- 2016-01-21 US US15/544,710 patent/US10435450B2/en active Active
- 2016-01-21 CA CA2973300A patent/CA2973300C/en active Active
- 2016-01-21 DK DK16701312.7T patent/DK3247719T3/da active
- 2016-01-21 EP EP16701312.7A patent/EP3247719B1/en active Active
- 2016-01-21 PL PL16701312T patent/PL3247719T3/pl unknown
- 2016-01-21 WO PCT/EP2016/051267 patent/WO2016116577A1/en active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002088354A1 (en) * | 2001-04-27 | 2002-11-07 | Medical Research Council | The nk1 fragment of hepatocyte growth factor/scatter factor (hgf/sf) and variants thereof, and their use |
US20060003931A1 (en) * | 2004-05-06 | 2006-01-05 | Genentech, Inc. | Crystal structure of the hepatocyte growth factor and methods of use |
CN101356189A (zh) * | 2005-11-10 | 2009-01-28 | 受体生物公司 | 肝细胞生长因子内含子融合蛋白 |
US20090215686A1 (en) * | 2007-03-05 | 2009-08-27 | Huaqiang Eric Xu | Nk1-based polypeptides and related methods |
WO2011116396A2 (en) * | 2010-03-19 | 2011-09-22 | The Board Of Trustees Of The Leland Stanford Junior University | Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists |
Non-Patent Citations (3)
Title |
---|
CASSIE J. LIU等: "An engineered dimeric fragment of hepatocyte growth factor is a potent c-MET agonist", 《FEBS LETT》 * |
H SAKATA等: "Heparin Binding and Oligomerization of Hepatocyte Growth factor/scatter Factor Isoforms. Heparan Sulfate Glycosaminoglycan Requirement for Met Binding and Signaling", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
赵亚力等: "干细胞生长因子及受体传递的调节与内吞作用", 《生物技术通讯》 * |
Also Published As
Publication number | Publication date |
---|---|
PL3247719T3 (pl) | 2020-07-13 |
HUE047148T2 (hu) | 2020-04-28 |
EP3247719A1 (en) | 2017-11-29 |
EP3247719B1 (en) | 2019-09-25 |
DK3247719T3 (da) | 2019-12-16 |
WO2016116577A1 (en) | 2016-07-28 |
JP2018508197A (ja) | 2018-03-29 |
PT3247719T (pt) | 2019-12-23 |
JP6944875B2 (ja) | 2021-10-06 |
CN107531770B (zh) | 2021-10-08 |
US10435450B2 (en) | 2019-10-08 |
ES2761852T3 (es) | 2020-05-21 |
CA2973300A1 (en) | 2016-07-28 |
US20170369545A1 (en) | 2017-12-28 |
CA2973300C (en) | 2024-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
FI121014B (fi) | Sinkkisormiproteiinijohdannaisia ja niiden menetelmiä | |
US8383575B2 (en) | (DI)barnase-barstar complexes | |
CN104736185B (zh) | 治疗Tau病变的方法 | |
KR101516023B1 (ko) | 눈물 리포칼린 돌연변이 단백질 및 이를 얻는 방법 | |
TWI259837B (en) | Specific binding molecules for scintigraphy, conjugates containing them and therapeutic method for treatment of angiogenesis | |
JPH05502880A (ja) | トランスロケーション領域および細胞結合領域を有するハイブリッド分子 | |
US20220162283A1 (en) | Methods of treating skin tissue damage | |
AU2004253835A1 (en) | Polypeptides having binding affinity for HER2 | |
US9765155B2 (en) | Covalent disulfide-linked diabodies and uses thereof | |
JP2001506967A (ja) | 治療およびインビボ診断における免疫グロブリンの使用の結果としての免疫グロブリン誘発毒性の抑制方法 | |
JPH08509460A (ja) | ターゲッティング用化合物 | |
WO1983000810A1 (en) | Selective carcinostatic agent | |
AU3433189A (en) | Mutant human angiogenin (angiogenesis factor with superior angiogenin activity) genes therefor and methods of expression | |
AU2013344253A1 (en) | Aprotinin-derived polypeptide-antibody conjugates | |
RU2426745C2 (ru) | Рекомбинантный химерный белок фактора ингибирования нейтрофилов и гиругена и содержащая его фармацевтическая композиция | |
JP6618539B2 (ja) | ソルターゼを使用した酵素媒介性のポリペプチドコンジュゲーションのための方法 | |
JPH10501813A (ja) | 腫瘍処置のための組成物および方法 | |
JPH06502847A (ja) | 植物毒素ジェロニンの蛋白質構造 | |
CN107531770B (zh) | Met受体激动蛋白 | |
JP5330826B2 (ja) | Tab分子 | |
JPH04503960A (ja) | 高発現宿主細胞系からの生物活性血小板由来成長因子の産生 | |
JPH07509698A (ja) | 後生動物寄生虫に対するワクチン | |
CN108264568A (zh) | 重组多肽、核酸分子及其组合物以及制造、使用其的方法 | |
JP2018531008A (ja) | ソルターゼaを利用してチオエステルを作製するための方法 | |
JP2018531008A6 (ja) | ソルターゼaを利用してチオエステルを作製するための方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |