Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-08-15
2004-08-10
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069400, C435S069510
Reexamination Certificate
active
06773899
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
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 operably linked to the T7 promoter, with bacteriophage &lgr; 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 &lgr; each carry at least one copy of the heterologous DNA.
2. Description of the Related Art
At present, genetic engineering methods allow creating microorganism strains capable of producing substantial amounts of various bioactive substances having important applications in medicine and industry. Typically, plasmid vectors into which a heterologous gene has been inserted are used to transform bacterial host cells. Different strains of
E. coli
are frequently used as recipient cells. Using such plasmid-dependent transformation methods,
E. coli
cells have been engineered to produce a variety of valuable human peptides and proteins, including insulin, &ggr;-interferon, a number of interleukins, superoxidedismutase, plasminogen activator, tumor necrosis factor, erythropoietin, etc. These substances are either already used in medical practice or undergoing different stages of clinical studies.
However, 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, polyethylemenin processing, illumination, sedimentation by ammonium sulfate, dialysis, and centrifugation (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.
A further complicating factor is that at relatively high levels of expression of the cloned genes, the eukaryotic proteins generated tend to accumulate in the cytoplasm of
E. coli
as insoluble aggregates, which are often associated with cell membranes. Consequently, the already difficult extraction and purification methods discussed above should be supplemented with additional technical procedures related to the extraction of the insoluble inclusion bodies. Usually, 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 M guanidine-HCl.
Often, the final stage of purification involves renaturation and reoxidation of the solubilized polypeptides, which is required to restore functional activity. Disulfide bonds, which are necessary for proper folding of the protein in its native conformation, should be reformed. Renaturation procedures, such as disulfide interchange, may use expensive and relatively toxic reagents, like glutathione, and oxidized 2-mercaptoethanol or dithiothreitol. Further, the final yield of bioactive genetically-engineered proteins may be relatively low. Moreover, the presence of even trace concentrations of the toxic reagents needed to solubilize and then re-establish secondary and tertiary protein structure may prohibit subsequent clinical application of the proteins. Thus, the generation of targeted protein in the form of insoluble inclusion bodies within the bacterial host cells not only complicates the production of recombinant proteins and results in diminished yield, but may also render the final protein unsuitable for clinical use (Fisher, B., Sumner, I., Goodenough, P. Biotech. and Bioeng. 41:3-13, 1993).
The technological difficulties associated with the extraction of proteins produced by the expression of heterologous genes from plasmid-transformed bacterial host cells may be overcome by infecting the transformed bacterial host cells with bacteriophage, whose lytic pathway results in lysis of the bearer cell. Thus, the desired protein may be simply released into the culture medium (Breeze A. S. GB 2 143 238A). Accordingly, Breeze disclosed a method of increasing the yield of enzyme produced in
E. coli
by infecting the bacterial cells with phage &lgr; carrying a temperature-sensitive mutation in cI to provide controlled lysis. The cl-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., Fritsch, E. F., Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, 1982, Cold Spring Harbor Laboratory Press). Bacteriophages carrying a temperature-sensitive mutation in cI are able to establish and maintain the lysogenic state as long as the cells are propagated at a temperature that allows the cI-gene product to repress transcription of phage genes necessary for lytic growth. Accordingly, the transformed bacterial host cells may be cultivated at 30° C., wherein the cI-mediated suppression of phage DNA transcription continues and the phage remains in the lysogenic 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 cI 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).
Although 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
E. coli
cells. Synthesis at the optimum temperature (37° C.) is not possible, since cells at temperatures exceeding 32° C. undergo lysis 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 min as disclosed by Breeze may activate proteases that destroy the targeted protein.
Auerbach et al. (U.S. Pat. No. 4,637,980) used a phage &lgr; DNA fragment for inducing lytic release of recombinant products. In that method, like Breeze, the temperature-sensitive mutation in &lgr; cI-gene product was used to provide temperature-dependent lysis of the bacterial host cells. The &lgr; DNA fragment in Auerbach maintained functional endolysin-encoding genes, N, Q, R and S, for producing lysozyme following inactivation of the cI repressor at 42° C. Most of the remaining phage genes were deleted; mutations in O and P genes prevented replication of the phage DNA. Consequently, the &lgr; DNA was not a fully functional phage, capable of modulating expression of the targeted gene. Moreover, the &lgr; DNA of Auerbach was not suitable for use as a vector for carrying targeted genes. Further, as discussed above, incubation of the bacterial host cells at 42° to 44° C. for 90-120 min as disclosed by Auerbach may activate proteases that destroy the targeted protein.
In addition to providing for the lytic release of intact protein from bacterial producer cells, bacteriophages have also been used as an alternative to bacterial plasmid-based vectors, for carrying heterologous DNA into host bacterial cells. (Murray, N. E. and Murray, K.,
Nature
251:476-481, 1974; Moir, A., Brammar, W. J.,
Molec. gen. Genet
. 149:87-99, 1976). Typically, 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.
175:77-87, 1979; Walter, F., Siegel, M., Malke, H., 1990, DD 276,694; Mory, Y., Revel, M., Chen, L., Sheldon, I.
Chernykh Svitlana I.
Kordyum Vitaliy A.
Slavchenko Iryna Yu.
Vozianov Oleksandr F.
Knobbe Martens Olson & Bear LLP
Mosher Mary E.
Phage Biotechnology Corporation
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