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  • 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
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  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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  • C12N2795/00—Bacteriophages
  • C12N2795/00011—Details
  • C12N2795/10011—Details dsDNA Bacteriophages
  • C12N2795/10111—Myoviridae
  • C12N2795/10141—Use of virus, viral particle or viral elements as a vector
  • C12N2795/10143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• This invention relates to recombinant DNA technology and more particularly to a new method for enhancing the production of heterologous proteins in bacterial host cells.
• the disclosed method involves infecting host cells, which contain plasmid encoding the gene of interest, with bacteriophage ⁇ to induce lysis of the bacterial host cells.
• Super-production may be achieved in selected host cells either when the plasmid alone carries at least one copy of the heterologous DNA or when both plasmid and phage ⁇ each carry at least one copy of the heterologous DNA.
• plasmid vectors into which a heterologous gene has been inserted are used to transform bacterial host cells.
• Different strains of £ colt are frequently used as recipient cells.
• £ co/i cells have been engineered to produce a variety of valuable human peptides and proteins, including insulin, ⁇ -interfero ⁇ , a number of interleukins, superoxidedismutase, plasminoge ⁇ activator, tumor necrosis factor, erythropoietin, etc.
• the plasmid method has serious disadvantages. It is technologically complicated, since the desired product has to be extracted from bacterial cells after biosynthesis, which is a multi-stage process. For example, interferon extraction involves disintegration of cells, buffer extraction, polyethyle ⁇ e-imine processing, clarification, precipitation by ammonium sulfate, dialysis, and ce ⁇ t ⁇ fugation (Goeddel, EP 0043980). The necessity for such extraction and purification steps not only complicates production technology of the recombinant product, but also results in substantial losses, especially during large-scale industrial production.
• the insoluble proteins are solubilized using ionic detergents, such as SDS or laurylsarcosine, at increased temperatures or in the presence of denaturants, such as 8 M urea or 6 8 guanidine-HCI.
• Renaturation procedures such as disulfide interchange, may use expensive and relatively toxic reagents, like glutathione, and oxidized 2 mercaptoethanol or dithiothreitol.
• the final yield of bioactive genetically engineered proteins may be relatively low.
• the presence of even trace concentrations of the toxic reagents needed to solubihze and then re establish secondary and tertiary protein structure may prohibit subsequent clinical application of the proteins.
• Breeze disclosed a method of increasing the yield of enzyme produced in £ coli by infecting the bacterial cells with phage ⁇ carrying a temperature sensitive mutation in c ⁇ to provide controlled lysis.
• the gene product is a repressor of early transcription and consequently blocks transcription of the late region of the phage DNA, which is required for head and tail assembly and cell lysis (Mantiatis, T., Fntsch, E.F., Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, 1982, Cold Spring Harbor Laboratory Press).
• Bactenophages carrying a temperature-sensitive mutation in c ⁇ are able to establish and maintain the lysogemc state as long as the cells are propagated at a temperature that allows the -gene product to repress transcription of phage genes necessary for lytic growth.
• the transformed bacterial host cells may be cultivated at 30° C, wherein the -mediated suppression of phage DNA transcription continues and the phage remains in the lysogemc state, until the stage of maximum ferment production is reached. Subsequently, the culture temperature may be increased to 42° C for 30 minutes in order to inactivate the el repressor and permit the phage to begin its lytic development.
• the host cells may then be incubated for 2 3 hours at 30° C to allow complete lysis and release of the enzyme (Breeze A.S. GB 2 143 238A).
• Breeze teaches release of proteins from bacterial producer cells, it requires cultivating producers at temperatures not exceeding 30° C, which is not the optimum temperature for growth of £ coli cells. Synthesis at the optimum temperature (37° C) is not possible, since cells at temperatures exceeding 32° C undergo iysis before reaching the stage of maximum ferment accumulation due to the development of temperature-sensitive lytic prophage. Furthermore, incubation of the bacterial host cells at 42° C for 30 mm as disclosed by Breeze may activate proteases that destroy the targeted protein.
• Auerbach et al. U.S. Patent No. 4,637,980
• a phage ⁇ DNA fragment for inducing lytic release of recombinant products.
• the temperature-sensitive mutation in ⁇ c ⁇ gene product was used to provide temperature-dependent lysis of the bacterial host cells.
• the ⁇ DNA fragment in Auerbach maintained functional endolysin-encoding genes, N, Q, R and S, for producing lysozyme following inactivation of the c ⁇ repressor at
• ⁇ DNA was not a fully functional phage, capable of modulating expression of the targeted gene.
• the ⁇ DNA of Auerbach was not suitable for use as a vector for carrying targeted genes.
• bactenophages have also been used as an alternative to bacterial plasmid-based vectors, for carrying heterologous DNA into host bacterial cells.
• bactenophages have also been used as an alternative to bacterial plasmid-based vectors, for carrying heterologous DNA into host bacterial cells.
• amplification of genes and their products is achieved during lytic growth of the phage, wherein the phage genome is integrated into the bacterial host DNA (Panasenko, S.M., Cameron, J.R., Davis, R.V., Lehman, L.R., Science 196:188-189, 1977; Murray, N.E. and Kelley, W.S., Molec. gen. Genet.
• ⁇ vectors have been used primarily for gene cloning. Once cloned, the genes are transferred to plasmid vectors for more effective expression. For example, when £ c ⁇ // ⁇ s infected by phage ⁇ Charon 4A C15, containing the human ⁇ - interferon gene, the quantity of interferon in cell lysate constituted 7-8 x 10 6 units/liter. After the DNA fragment bearing targeted gene was recloned from phage to plasmid, ⁇ -interferon yield increased to 1 x 10 8 units/liter (Moir, A., Brammar, W.J., Molec. gen. Genet. 149:87-99, 1976).
• the yield of DNA gase 1 in lysogemc cultures containing ⁇ gt4l ⁇ gS prophage, with amber-mutation in the S gene was five times greater than the yield of DNA ligase 1 in lysogemc cultures containing ⁇ gt4l ⁇ g prophage without the amber-mutation (Panasenko, S.M., Cameron, J.R., Davis, R.V., Lehman, L.R., Science 196:188-189, 1977).
• the phage ⁇ S protein is required for lysis; therefore S mutants accumulate large numbers of intracellular progeny phage particles, as well as the targeted protein, without I ⁇ sing the host cells (Mantiatis, T., Fntsch, E.F., Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, 1982, Cold Spring Harbor Laboratory Press).
• the present invention relates to a method for producing a soluble and biologically active protein.
• the method comprises the steps of transforming an £ c ⁇ // host ceil with a plasmid having at least one copy of an expressible gene encoding the protein.
• the host ceil is infected with a bacteriophage ⁇ capable of growth without lysis at a permissive temperature and also capable of mediating lysis at a restrictive temperature.
• the host cells are cultivated at the permissive temperature until a level of production of the protein is at least 100 ⁇ g/ml.
• the host cells are then lysed to release the soluble and biologically active protein by cultivating the host cell at the restrictive temperature.
• the bacteriophage ⁇ has a temperature-sensitive mutation, preferably, the temperature-sensitive mutation is cl 857 .
• the restrictive temperature is greater than 32° C.
• the permissive temperature is less than about 32° C.
• the bacteriophage ⁇ has a mutation in at least one gene, the expression of which at the restrictive temperature mediates lysis of the host cell.
• the at least one gene may be selected from the group consisting of N, D and R.
• the £ coli host cell produces a suppressor for the repair of amber mutations.
• the £ coli host cell lacks a suppressor for the repair of amber-mutations.
• the infecting bacteriophage ⁇ may be provided at a multiplicity of infection in a range of about 1 to about 100. More preferable, the infecting bacteriophage ⁇ is provided at a multiplicity of infection in a range of about 10 to about 25, wherein lysis of the host cell at the restrictive temperature is delayed at higher multiplicities of infection relative to lower multiplicities of infection.
• the bacteriophage ⁇ contains at least one copy of the expressible gene encoding the protein
• the present invention is also related to an £ coli host cell, which is adapted to produce a soluble and biologically active protein at a concentration of at least 100 ⁇ g/ml
• the host cell has a plasmid with at least one copy of an expressible heterologous gene encoding the protein.
• the host cell also contains a bacteriophage ⁇ capable of growth without lysis at a permissive temperature and also capable of mediating lysis at a restrictive temperature.
• the bacteriophage ⁇ contained in the £ coli host cell of the present invention may have a temperature sensitive mutation, preferably, the temperature sensitive mutation is d 857 .
• the bacteriophage ⁇ contained in the £ coli host cell may also have a mutation in at least one gene, the expression of which at the restrictive temperature mediates lysis of the host cell.
• the at least one gene may be selected from the group consisting of N, Q and R.
• the £ coli host cell contains a bacteriophage ⁇ with d 857 , Q am l 17 and R am 54 mutations.
• the bacteriophage ⁇ has at least one copy of an expressible heterologous gene encoding the protein.
• the £ coli host cell may lack a suppressor for repairing amber-mutations. In another mode, the £ coli host cell may be recA deficient.
• the expressible heterologous gene encodes human alpha 2b interferon.
• Bacteriophage ⁇ is useful as a vector because more than 40% of the viral genome is not essential for lytic growth.
• This area of the ⁇ genome, located in the central region of the ⁇ DNA, between genes J and N, may be replaced by heterologous DNA encoding a desired product. That region is transcribed early during infection.
• special conditions for the phage's development must be provided to ensure proper replication. Further, transcription of the early area, containing the targeted gene, should be fostered, while transcription of the later genes, involved in cell lysis, should be decelerated.
• Deceleration of late transcription may be accomplished by: (1) mutations of phage genome that block expression of the later genes (2) increased multiplicity of infection, and/or (3) cultivation of the infected bacterial cells at a reduced temperature.
• phage causes a profound rearrangement of all macromolecular synthesis in the bacterial host cells.
• phages may increase the copying of the targeted gene, and consequently, increase the output of desired product.
• phage ⁇ with amber-mutations that delay bacterial lysis are provided in a strain of £ coli, designated Su°, which lacks the suppressor responsible for correcting amber-mutations in phage ⁇ .
• Su° clones are selected from the wild-type Su * population.
• a selection marker is inserted into the phage DNA, e.g., tetracycline or ampicillin resistance.
• Phage Lvsate - Lysogemc culture was grown in broth at 28° C under intense aeration to a density of 2 x 10 8 cells/ml followed by incubation at 43° C for 20 mm. Then it was kept at 37° C under intense aeration. Cells were lysed in 60-80 mm and phage was released into the cultural medium. Phage titer was estimated by a conventional two-layer technique. As a rule, 2 x 10'° PFU/ml of phage lysate were obtained.
• phage ⁇ N' mutant is not able to lyse the host cells and is present in cells in the form of extremely unstable plasmids. If the host cells contain suppressor, the amber-mutation is phenotypicali ⁇ corrected, the N protein is synthesized and the phage can develop lytically. This difference in the viability of Su * and Su° cells, infected by ⁇ N', is used as a basis for selection of spontaneously appearing Su° reverta ⁇ ts from the £ coli Su * cell population. Phage ⁇ with an inserted plasmid that introduced the ampicillin and tetracycline resistance markers into cells was used to prevent the nonlysing Su° cells from masking the search for mutants. The phage also contains ts mutation in the repressor gene that permits lytic development of such phage resulting in cell lysis.
• Su° derivatives of the parent cultures are obtained by curing the cells from the phage. The method can be subdivided into several stages.
• the culture £ c ⁇ // Su * was grown on the M9 medium with maltose at 37° C under intense agitation to a density of 1 2 x 10 8 cells/ml.
• the cells were infected with phage ⁇ bla N' at a multiplicity of 5 10 particles per cell and incubated for 20 mm at 20° C. Under given conditions, the infection efficiency is about 100%, in addition to the bulk of Su * cells, the phage also infects single Su° cells.
• Isogenic suppressor-free derivatives of the parent £ coli Su * strains are clones, on which phage ⁇ bla N' did not form plaques, phage ⁇ d S57 Q amll7 R am54 produced 1 3 x 10 5 PFU/ml, and phage ⁇ d 857 without mutations in genes Q and R produced 1 x 10'° PFU/ml.
• Su° revertants of £ coli K 802 Su * were obtained. Based on the cell number at the moment of infection and the number of Su° revertants among them, the frequency of occurrence of suppressor-free ceils was about 3 x 10 7 .
• Example 1 Increased Synthesis of ⁇ Lactamase in £ coli Transformed with pBR322 Carrying the ⁇ Lactamase Gene
• the ⁇ treated cultures were incubated for 15 mm at 37° C to inactivate the d repressor, and then for 19 hr at 28° C.
• the control cultures were incubated at 37° C for the entire period, ⁇ Lactamase activity was determined by lodomet ⁇ c assay as described by Chaykovakaya, S.M. and Venkina, T.G. Antibiotics 7(51:453-456, 1962.
• a unit of activity is defined as the minimum guantity of ferment necessary to inactivate 1 x 10 7 M penicillin (60 units) in 1 hr at 37° C, pH 6.8 7.0.
• the second portion was infected with phage ⁇ cl 587 bla Q am ll7 R a ⁇ l 54 at a multiplicity of about 10 phage bodies per 1 bacterial cell and cultivated for 2.5 3 hr at 37° C, and then for an additional 14 hr at 28° C ⁇ Lactamase activity was measured by the lodometric method.
• the results, shown in Table 2 (below), are expressed in units, as defined above for Table 1.
• Phage ⁇ d 587 bla Q am I17 R am 54 was prepared from lysogemc cultures maintained at 28° C in Ammopeptid medium. When the bacterial cell density reached about 1 x 10 s cells/ml, the cells were warmed for 20 minutes at 43° C in order to inactivate the d repressor. Consequently, the prophage is excised from the bacterial genome and begins its lytic development. After 50 mm, the cells underwent lysis, releasing 100 200 bodies each.
• bacterial cells which were transformed with both plasmid containing the targeted gene and phage carrying the same gene produced about 10 times more recombinant protein ( ⁇ -lactamase) than bacterial cells transformed with phage alone, and over 100 times more ⁇ -lactamase than bacterial cells transformed by plasmid alone.
• the temperature was then decreased to 37° C and the bacterial cells underwent lysis, with phages being formed at 1-2 x 10'° PFU/ml.
• 10 liters of phage lysate containing about 1 x 10'° phage bodies ( ⁇ d 857 plac5 Q am ll7 R am 54 ) per ml, were added to 40 liters of a suspension of E coli Ca 77 Su° transformed by plasmid pZ56 at a density of about 1 x 10 8 cells/ml in LB medium.
• the multiplicity of infection was 25, i.e., there were 25 phage bodies per bacterial cell.
• ⁇ -galactosidase constituted 1.9 g per liter of culture medium.
• the activity of ⁇ -galactosidase was calculated by the method of Miller (Miller, J.H., EXPERIMENTS IN MOLECULAR GENETICS, 1972, Cold Spring Harbor Laboratory Press). A unit of activity was calculated as the minimum quantity of ferment required to hydrolyze 1 ⁇ M ortho-mtrophenyl- ⁇ -D galactoside to ortho nitrophenol per mm at 30° C, pH 7.0.
• Strain £ Coli SG 20050 was transformed by a plasmid bearing two copies of the human interferon alpha-2b gene (plF-14) by standard methods.
• the transformant cells were grown up in 80 liters of LB medium at 37° C with intensive aeration to a density of 2 x 10 8 cells/ml.
• the culture was divided into two portions. The first was not infected with phage. The second was infected with phage ⁇ lysate harvested from £ co/i K 802/ ⁇ d 857 Q am ]17 R am 54 at a multiplicity of 10 phage bodies per bacterial cell.
• the control cells were incubated for 19 hr at 37° C and the phage infected cells were incubated for 19 hr at 21 ° C.
• Interferon production in both control and phage-infected cultures was about 20% of the total cellular protein.
• the interferon in control cells was associated at least in part with insoluble inclusion bodies.
• the interferon activity was 4 x 10 10 lU/ ter (200 mg/liter).
• Embryotoxic studies of recombinant interferon were conducted in pregnant hamadryad baboon females. Daily intra-muscular doses during organogenesis (20 ,h to 50 , ⁇ days of pregnancy) caused defects in embryo development leading to miscarriage or stillbirth. Similar results were obtained for recombinant interferon analogs, and most probably could be explained by a powerful antiproliferative action of interferons It is possible that the miscarriage may be attributed to a "cancellation" of immunologic tolerance of maternal organism towards the fetus, caused by immuno modulating action of the protein.
• Recombinant interferon was also studied in Ukrainian clinics. Based on these clinical studies, the recombinant interferon was shown to be useful in the treatment of a variety of human diseases and conditions. For example, recombinant interferon was effective in treating acute and chronic hepatitis B, acute viral, bacterial and mixed infections, acute and chronic septic diseases, herpetic infections, herpes zoster, papillomatosis of larynx, multiple sclerosis, and various cancers, including melanoma, renal ceil carcinoma, bladder carcinoma, ovarian carcinoma, breast cancer, Kaposi's sarcoma and myeloma. The contraindications in human clinical applications were prolonged (several months) use at high doses, allergy and pregnancy. The possible side effects noted were small and transitory "flu-like" symptoms and at prolonged regimens, leuko and trombocytope ⁇ ia were marked.

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