WO1987002702A1 - Virus lytiques utilises comme vecteurs d'expression, cellule hote les contenant et procede de production de proteines - Google Patents

Virus lytiques utilises comme vecteurs d'expression, cellule hote les contenant et procede de production de proteines Download PDF

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Publication number
WO1987002702A1
WO1987002702A1 PCT/SE1986/000477 SE8600477W WO8702702A1 WO 1987002702 A1 WO1987002702 A1 WO 1987002702A1 SE 8600477 W SE8600477 W SE 8600477W WO 8702702 A1 WO8702702 A1 WO 8702702A1
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gene
phage
virus
host cell
chromosome
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PCT/SE1986/000477
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English (en)
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Alexander Ulrich Von Gabain
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Kabigen Ab
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • Lytic viruses as expression vectors, host cell containing same and process for protein production
  • the present invention relates to lytic viruses, such as bacterial phages.
  • the invention also covers host cells infected by such viruses as well as a process for using such viruses for the preparation of a heterospecific protein in a host cell.
  • the invention has for an object to provide new biological techniques enabling integration of episomes in the chromosome of a lytic virus and maintaining the recombinant characteristic of the virus through its infectious lifecycle.
  • E.coli phage T5 which is a known bacteriophage.
  • the main object of the present invention is to provide a lytic virus which in its chromosome has inserted an alien production gene, the recombinant virus being stable through its infectious lifecycle. Such production gene is preferably positioned in a non-essential region of the chromosome of the virus.
  • the virus of the invention is preferably a bacteriophage, such as an E.coli phage T5.
  • the phage is preferably selected from T5+ or its derived stable and autonomously viable deletion mutants, such as T5 sto.
  • the production gene has preferably been introduced into the chromosome of the virus by so called homologous reciprocal recombination.
  • the invention also covers host cells infected with such lytic recombinant virus.
  • the host cell is preferably an E.coli.
  • Another object of the invention is to provide for a process for preparing a heterospecific protein which can be recovered in an expedient manner.
  • a process for preparing a protein by expression in a host cell comprises the steps: a) introducing an alien production gene in the chromosome of a lytic virus; b) infecting a host cell by introducing the resulting recombinant virus into same; c) allowing expression of the recombinant protein in said host cell; and d) recovering the protein thus produced.
  • the production gene under step a) is introduced by homologous reciprocal recombination.
  • the gene is preferably introduced in a non-essential region of the phage chromosome, and put under control of phage promotors.
  • E.coli bacteria as host cells to be infected by the recombinant phage.
  • phagebased production system taking advantage of three attractive properties of the T5 system, namely the immediate and complete shut- off of host protein synthesis, the degradation of the host chromosome and making use of promotors of extreme signal strength.
  • the techniques of this invention can be applied in different ways, among which the following two applications are of major importance.
  • the invention enables facile production of recombinant heterospecific proteins by introducing the desired production gene in the virulent phage used.
  • a system based on the lytic phage T5 offers a number of novel features concerning the expression of alien genes, that could be applied in biotechnology:
  • the complete shut-off of the host protein synthesis right after the infection limits the expression of infected cells to phage derived and recombinant proteins.
  • the degradation of the host chromosome to acid solubility reduces problems of DNA viscosity found, when recombinant proteins are purified from E.coli (Fish, N.M. & Lilly, M.D. (1984) Biotechnology 2, 623-627).
  • a set of deletion mutants and plasmids designed fo ⁇ insertion could be constructed that are suitable for different sized inserts and optimal expression concerning the juxtaposition of promotors and inserts.
  • plasmids designed for insertion could be furnished with an indicator gene, like the one encoding S-galactosidase, allowing to identify recombinant phages by a colorimetric plaque test described for other phages (Messing, J., Gronenborn, B., Muller-Hill, B. & Hofschneider, P.M. (1977) Proc.Natl.Acad.Sci. USA 3642-3646).
  • a further modification of this strategy is the use of T5-mutants, defective in the "early"-”late” switch, which can be amplified on suppres sor host strains. Those phages give rise to abortive infections stagnating at the "early” stage when non permissive cells are infected (McCorquodale, D . J . (1975) CRC Critical Reviews in Microbiology 4, 213-234).
  • the advantage of an abortive infection system is the long period expression of "early” proteins without entering the stage of phage DNA replication and lysis of the cells.
  • such a system can be applied to analyze recombinant gene products without interference of cellular protein synthesis, as it is done by using mini cells (Frazer, A.C. & Curtiss III, R. (1975) Curr.Top.Microbiol.Immunol. 69, 1-84) or maxi cells (Sancar, A., hack, A.M. & Rupp, D. (1979) J. Bacteriol. 137, 692-693).
  • mini cells Frazer, A.C. & Curtiss III, R. (1975) Curr.Top.Microbiol.Immunol. 69, 1-84
  • maxi cells Small Car, A., hack, A.M. & Rupp, D. (1979) J. Bacteriol. 137, 692-693.
  • the approach is based on inserting the genes of interest in phages, as it has been demonstrated for the CAT gene, and analyzing the short term radio-labeled extracts of in
  • Fig. 1 shows an autoradiogram of BNA-blotting
  • Fig. 2 shows by autoradiogram the activity of chloramphenicole acetyl transferase in infected cells
  • Fig. 3 is a model of the physical structure of the T5 phage.
  • Fig. 4 is a map of one integration plasmid used.
  • Fig. 5 is a map of another integration plasmid used.
  • T5 has a number of unique properties making the organism attractive to study the integration of episomes in its chromosome.
  • the phage is organized in a linear double stranded DNA molecule exhibiting a length of 121284 basepairs (Rhoades, M. (1982) J.Virol. 43, 566-573).
  • Stable deletions in a non-essential region of the chromosome may allow insertion of genetic material up to a size of 14,000 base ⁇ airs.
  • the yield of phage-directed biosynthesis during the infection is remarkable: with a burst size of about 200 phage particles per cell the infected cells replicate within the infectious cycle of 45 minutes about 2.4 . 10 7 base pairs.
  • pPL 1 is derivative of pPL 603 containing a Chlorampehnicol-acetyl-transferase (CAT) gene from B.Pumilis (see Williams, D.M., Duvall, E.J. & Lovett, P.S. (1981) J. Bacteriol. 146, 1162-1165); it was donated by L. Rutberg.
  • CAT Chlorampehnicol-acetyl-transferase
  • the restriction fragment Hindlll 0 derived from the T5 chomo- some was inserted in the unique Hindlll site of pBR 322 or pACYC 177; the constructs were named pBRO and pACYCO, respectively.
  • the distance between the EcoRI site assymetrically mapping in the phage derived insert and the next EcoRI site on pBR322 see Rodriquez, R.L.F., Bolivar, R.J., Greene, M.C., Betlach, H.L., Heynecker, H.L., Boyer. H.W., Crosa, J.H. & Falkow, S. (1977) Gene 2, 95-113) and the next Xhol site on pACYC 177 (see Chang, A.C.Y. & Cohen, S.N. (1978)
  • J. Bacteriol. 134, 1141-1156) were determined in order to figure out the orientation of the inserts in the two vectors (see also Figure 5).
  • pBR ⁇ O was derived from pBRO by removing the 249 basepair EcoRI fragment spanning from the phage insert into the pBR 322 vector.
  • pPR ⁇ OCAT finally was constructed by inserting the Bglll - BamHI fragment isolated from pPL 1 in the unique BamHI site of pPR ⁇ O as described below in the result section and as indicated in Figure 5. Growth and Purification of Phages. T5 + and T5 sto were grown as described by Bujard, H. & Hendrickson H.E. (1973) Europ. J.Biochem. 33, 517-528.
  • Recombinant phages were cultivated the same way but grown and purified in media, solutions and buffers containing 10mM MgSO 4 , and 1mM Spermine (Labedan, F.T.B. & Legault-Demare, J. (1984) J.Virol. 50, 213-219.) CsCl gradients were performed in a VTi 50 rotor (Beckman), 33,000 rpm, 40 hrs at 20°C. The initial density was adjusted to 1.56 g/ml.
  • phages were first purified in a CsCl step gradient performed in a swing out rotor (SW 40) 30,000 rpm, 1hr at 20°C; the phages were overlayered on a preformed step gradient containing equal volume (2.5 ml) of the following densities: 1.5 g/ml, 1.0 g/ml, 0.5 g/ml.
  • a preformed step gradient containing equal volume (2.5 ml) of the following densities: 1.5 g/ml, 1.0 g/ml, 0.5 g/ml.
  • the phageparticles were incubated with DNasel (10ug/ml, Worthington) for 1 hr at 37°C prior to loading them on the CsCl step gradient; the incubation was performed in phagesuspensionbuffer (Bujard, H. & Hendrickson, H.E. (1973) Europ.J.Biochem. 33, 517-528) containing 10mM MgSO 4 .
  • Plaque lift and Blotting techniques The number of plaque forming units was determined with the soft agar technique as described by Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular Cloning edited by Cold Spring Harbor Laboratory 1982 for e.g. the ⁇ phage with the following modifications the soft agar contained always 2mM CaCl 2 and additionally 10mM MgCl2 and 1mM Spermine when recombinant phages were plated.
  • Plaque lifts were performed with Nitrocellulose filters following the procedure as it is described for recombinant ⁇ phages (loc.cit.); hybridization and washing of filters were persued according to the same protocol; the filters were finally exposed to X-ray film (Kodak X-OMAT) for periods of 2-24 hrs. Blotting of electrophoretically fractionated RNA or DNA samples was carried out following procedures previously described (loc.cit.). For all hybridizations plasmid UNA or in some cases isolated DNA fragments were nicktranslated using 32 P-labeled deoxyribonucleotid-triphosphates as radio- isotopes (loc.cit.).
  • Chloramphenicol acetyl transferase (CAT) assay Cells were synchronously infected with recombinant phages (multiplicity of infection (M.O.I) was two) containing the CAT gene integrated in the chromosome; aliquots of 2ml were withdrawn at several timepoints during the infectious cycle and probed for their content of CAT enzyme as described by Nilsson, G., Belasco, J.G., Cohen. S.N. & von Gabain, A. (1984) Nature 312, 75-77.
  • M.O.I multipleplicity of infection
  • Figure 1 shows the autoradiogram of a plaquehybridization.
  • Cells harboring pBR OCAT were infected with T5sto (see Materials and Methods and notes to table below). After lysis 2000 phages were plated on agar plates (Panel A); the plaques were analyzed by the plaque-lift technique using Nitrocellulose as described in Materials and Methods.
  • the filter was hybridized with radio- labeled pPL 1 and further treated as described by Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular Cloning edited by Cold Spring Harbor Laboratory 1982.
  • An X-ray film (Kodak X- OMAT) was exposed to the filter for 4 hrs and afterwards processed.
  • Figure 2 shows an autoradiogram of a DNA blotting according to Southern (see Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular Cloning edited by Cold Spring Harbor Laboratory 1982).
  • Cells harboring the plasmid pACYCO were infected with T5 sto the phages were harvested as described above and DNA was extracted and digested by the restriction endonucleases Hindlll and PstI as indicated.
  • As control DNA isolated from T5 sto was digested by both enzymes and DNA from pACYC 177 by PstI (see Chang, A.C.Y. & Cohen, S.N. (1978) J. Bacteriol. 134, 1141-1156).
  • FIG. 3 is an autoradiogram showing the activity of chloramphenicol acetyltransferase in recombinant T5 infected cells. 10 min after infection the different forms of mono- and diacetylated chloramphenicol appear. Cell extracts were made by taking aliquots of cells before infection 2, 4, 10 and 20 min after infection, and disrupted by lysozyme treatment and sonication. This cell extract was assayed by incubation with 14 C-labelled chloramphenicol and analysed by ascending TLC (see Nilsson, G., Belasco, J.G., Cohen, S.N.
  • Figure 4 is a model of the physical structure of
  • T5st(0) DNA (113 kb) with its "nickpattern”.
  • the map of the Hindlll restriction sites is shown below the physical map.
  • the region where the plasmid is integrated (by homologous recombination) is blown up, showing a physical map of the section of the T5 genome encoding the phage-specific stable RNAs (see Ksenzenko, V.N., Kamynina, T.P., Kazantsev, S.I., Shlyapnikov, M.G., Krykov, V.M. & Bayev, A.A.
  • Figure 5 is a map of the integration plasmid carrying a 906 bp EcoRI-Hind III fragment from T5.
  • the CAT-86 gene ob tained from pPL1 (Rutberg personal communication), a derivative of pPL 603 (see Williams , D.M., Duvall, E.J. & Lovett, P.S. (1981) J. Bacteriol. 146, 1162-1165), is inserted in the BamHI site.
  • the CAT gene is deprived of its promotor (loc.cit.).
  • Hindlll fragment 0 (1200 basepairs length) mapping in the non essential region of the T5 chromosome was inserted in the plasmids pBR322 (Rodriquez, R.L.F., Bolivar, P.J., Greene, M.C., Betlach, H.L., Heynecker, H.L., Boyer, H.W., Crosa, J.H. & Falkow, S. (1977) Gene 2, 95-113) and pACYC 177 (Chang, A.C.Y. & Cohen, S.N. (1978) J.Bacteriol. 134, 1141-1156) as described above.
  • E.coli containing either the chimeric plasmid pACYCO or pBRO and its derivatives were infected with T5 st.o, a deletion mutant of the wild-type phage missing 8570 base pairs in the deletable region next to the region spanning the Hindlll fragment 0.
  • the descending phages were plated on agar plates in order to determine the number of plaque forming units.
  • the indicator strain did not contain the respective plasmids. Plates containing between 10 2 and 10 4 plaques were submitted to plaquelift (see Maniatis, T., Fritsch, E.F.
  • Phages derived from plaques giving positive signal were expanded in fluid cultures as described above. After purifying them over a CsCl-step gradient, DNA was extracted and dissected in parallel with several restriction endonucleases. The DNA fragment were fractionated on agarose gels and subsequently transferred on to nitrocellulose. The filters were probed with the respective plasmids not carrying the phage insert and with the Hindlll fragment 0.
  • Figure 2 shows the analysis of the recombinant phages obtained when E.coli was infected carrying e.g.
  • Hindlll digest disclosed an unique fragment (4.1 kb) lightening up exclusively with the plasmid-specific probe, whereas the PstI digest disclosed three fragments (20 kb, 6.8 kb, and 5.2 kb, respectively) hybridizing to both probes.
  • the result is compatible with a multiple insertion of the plasmid by homologous recombination:
  • the Hindlll fragment corresponds to the excised pACYC 0 ( Figures 2 and 4), whereas the smallest PstI fragment ( Figures 2 and 4) reflects the repeat of pACYC 177 and Hindlll 0, which is excised when plasmid and the phage fragment are arranged in duplications as a consequence of multiple insertions, the two largest PstI fragments (see Figure 2) correspond exactly to the predicted sizes expected when the distance to adjacent PstI sites in the phage chromosome is calculated (see Figure 4).
  • the phages were further characterized by fractionating recombinant phages and their predecessors on CsCl density gradients as described above. Typically two bands were observed, the upper and corresponded to a boyant density of 1.54 g/cm 3 we found for T5sto, whereas the lower one disclosed a boyant density of 1.56 g/cm 3 when e.g. pBR ⁇ O CAT (see Figure
  • CAT Chloroamphenicol transferase
  • E.coli cells MM 294 were synchronously infected with recombinant phages and the CAT activity was monitored during the course of the infections cycle by analyzing aliquots of infected cells as described previously (see Nilsson, G., Belasco, J.G., Cohen, S.N. & von Gabain, A. (1984) Nature 312, 75-77). The results are shown in Figure 3. Expression is not detectable before the "early" stage of infection, namely after about 10 minutes, and continues until the "late” stage of infection.
  • the phages were further amplified and then used to synchronously infect E.coli cells.
  • the multiplicity of infections was 5 phages per bacterial cell.
  • Aliquots of cells (2.5 x 10 9 ) were withdrawn from the culture before and after infection at indicated times (see the Table below) and the ⁇ -galatosidase content was determined using an enzyme assay (reference). Enzyme activity was detectable 10 minutes after infection and steadily increased until the lysis of the cells. It should be noted that the fraction of recombinant phages was not determined for the phagestock used in the experiment.
  • the experiment shows the general applicability of a T5 based expression system and furthermore the approach made it possible to identify recombinant phages, as blue peagues, in a colorimetric assa .
  • genes than the CAT gene as described above can be introduced in the chromosome of a suitable lytic virus to express other proteins of commercial interest.

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Abstract

Virus lytique dont le chromosome renferme un gène inséré pour produire des protéines hétéro-spécifiques; cellule hôte infectée par ledit virus lytique; et procédé de préparation d'une protéine hétéro-spécifique par expression dans une cellule haute dans laquelle on a introduit ledit virus lytique. Une cellule haute préférée est E.coli et des virus lytiques préférés sont les phages T5, T5+ et T5 sto.
PCT/SE1986/000477 1985-10-24 1986-10-15 Virus lytiques utilises comme vecteurs d'expression, cellule hote les contenant et procede de production de proteines WO1987002702A1 (fr)

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SE8505027A SE8505027D0 (sv) 1985-10-24 1985-10-24 A lytic virulent phage, a hostcell containing same and a process for protein production
SE8505027-6 1985-10-24

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EP0294595A2 (fr) * 1987-05-08 1988-12-14 Enzo Biochem, Inc. Composition polynucléotidique et méthode pour sa production
WO2000024873A1 (fr) * 1998-10-28 2000-05-04 Genentech, Inc. Procede de recuperation de polypeptides heterologues a partir de cellules bacteriennes
US8821855B2 (en) 2005-01-10 2014-09-02 Omnilytics, Inc Methods for isolating phage and for controlling microorganism populations with the phage
WO2022063986A3 (fr) * 2020-09-26 2022-06-16 Snipr Biome Aps Virus synthétiques
US11400110B2 (en) 2015-05-06 2022-08-02 Snipr Technologies Limited Altering microbial populations and modifying microbiota

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GB2105344A (en) * 1981-08-31 1983-03-23 Genentech Inc Preparation of polypeptides in vertebrate cell culture
US4415660A (en) * 1978-02-27 1983-11-15 President And Fellows Of Harvard College Method of making a cloning vector
US4460689A (en) * 1982-04-15 1984-07-17 Merck & Co., Inc. DNA Cloning vector TG1, derivatives, and processes of making
US4469791A (en) * 1980-02-15 1984-09-04 Cpc International Inc. Genetically engineered microorganisms for massive production of amylolytic enzymes and process for preparing same
US4495280A (en) * 1981-05-20 1985-01-22 The Board Of Trustees Of The Leland Stanford Jr. University Cloned high signal strength promoters
US4506013A (en) * 1980-10-03 1985-03-19 Eli Lilly And Company Stabilizing and selecting recombinant DNA host cells
US4508896A (en) * 1975-05-30 1985-04-02 Texaco Development Corporation Process for the simultaneous production of 2-(2-aminoalkoxy)alkanol and morpholine
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US4508896A (en) * 1975-05-30 1985-04-02 Texaco Development Corporation Process for the simultaneous production of 2-(2-aminoalkoxy)alkanol and morpholine
US4415660A (en) * 1978-02-27 1983-11-15 President And Fellows Of Harvard College Method of making a cloning vector
US4469791A (en) * 1980-02-15 1984-09-04 Cpc International Inc. Genetically engineered microorganisms for massive production of amylolytic enzymes and process for preparing same
US4506013A (en) * 1980-10-03 1985-03-19 Eli Lilly And Company Stabilizing and selecting recombinant DNA host cells
US4495280A (en) * 1981-05-20 1985-01-22 The Board Of Trustees Of The Leland Stanford Jr. University Cloned high signal strength promoters
GB2105344A (en) * 1981-08-31 1983-03-23 Genentech Inc Preparation of polypeptides in vertebrate cell culture
US4460689A (en) * 1982-04-15 1984-07-17 Merck & Co., Inc. DNA Cloning vector TG1, derivatives, and processes of making
EP0176170A1 (fr) * 1984-06-04 1986-04-02 Pasteur Merieux Serums Et Vaccins Virus Herpès simplex à titre de vecteur

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Eukaryotic Viral Vectors (Conf.), 1981, p 55-60, published 1982, Ed. GLUZMAN Y. (LIU C.-C. et al.): "Expression of Hepatitis B Surface Antigen Using Lytic and Nonlytic SV-40 Based Vectors". *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0294595A2 (fr) * 1987-05-08 1988-12-14 Enzo Biochem, Inc. Composition polynucléotidique et méthode pour sa production
EP0294595A3 (en) * 1987-05-08 1989-12-06 Enzo Biochem, Inc. Polynucleotide composition and method for its production
EP1323820A3 (fr) * 1998-10-28 2003-07-09 Genentech Inc. Procédé de récupération de polypeptides hétérologues à partir de cellules bactériennes
US6180367B1 (en) 1998-10-28 2001-01-30 Genentech, Inc. Process for bacterial production of polypeptides
US6258560B1 (en) 1998-10-28 2001-07-10 Genentech, Inc. Process for bacterial production of polypeptides
EP1323820A2 (fr) * 1998-10-28 2003-07-02 Genentech Inc. Procédé de récupération de polypeptides hétérologues à partir de cellules bactériennes
WO2000024873A1 (fr) * 1998-10-28 2000-05-04 Genentech, Inc. Procede de recuperation de polypeptides heterologues a partir de cellules bacteriennes
US8821855B2 (en) 2005-01-10 2014-09-02 Omnilytics, Inc Methods for isolating phage and for controlling microorganism populations with the phage
US11400110B2 (en) 2015-05-06 2022-08-02 Snipr Technologies Limited Altering microbial populations and modifying microbiota
US11517582B2 (en) 2015-05-06 2022-12-06 Snipr Technologies Limited Altering microbial populations and modifying microbiota
US11547716B2 (en) 2015-05-06 2023-01-10 Snipr Technologies Limited Altering microbial populations and modifying microbiota
US11612617B2 (en) 2015-05-06 2023-03-28 Snipr Technologies Limited Altering microbial populations and modifying microbiota
US11642363B2 (en) 2015-05-06 2023-05-09 Snipr Technologies Limited Altering microbial populations and modifying microbiota
US11844760B2 (en) 2015-05-06 2023-12-19 Snipr Technologies Limited Altering microbial populations and modifying microbiota
WO2022063986A3 (fr) * 2020-09-26 2022-06-16 Snipr Biome Aps Virus synthétiques

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