Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-03-13
2001-09-25
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S320100, C536S023700
Reexamination Certificate
active
06294372
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to replication genes and gene products identified in small cryptic plasmids, and more specifically, to methods for constructing controlled-replication plasmid vectors which are not lethal to the host and capable of very high levels of replication.
BACKGROUND OF THE INVENTION
The production of large quantities of proteins for use as therapeutics, additives, and other myriad applications remains a challenge. Large-scale fermentation is a commonly used method, but is expensive and difficult to maintain the required quality and consistency of product. When producing proteins in bacteria, vectors that have a high copy number are generally sought because the amount of protein is often directly proportional to gene dosage.
DNA vaccination, perhaps more precisely called DNA-mediated immunization, refers to the direct introduction into a living species of plasmid or even non-plasmid DNA (or RNA) which is able to cause expression of antigenic protein(s) or peptide(s) in the newly transfected cells. Presentation of DNA into tissues of the host species may be by needle injection, particle bombardment or even orally using various DNA formulations which may be either “naked” DNA, coated microparticles, or via liposomes or biodegradable microcapsules or microspheres. In any case relatively large quantities of purified plasmid DNA are required for production of these vaccines.
Runaway replication plasmid vectors have been developed for expression of genes in bacteria. These vectors are based on plasmids, such as RI in which an antisense RNA transcript is a negative regulator of repA translation and a product of the copB gene, is a repressor of transcription of repA. RepA protein, which functions in cis, is necessary and rate-limiting for replication. The copy number of the plasmid is determined by the relative levels of the antisense RNA and RepA mRNA. If the antisense RNA levels decrease and RepA mRNA increases, expansive plasmid replication results. This is achieved by defined temperature sensitive mutations in the plasmid regulatory region or by insertion of an additional inducible promoter (ex. &lgr; promoter) upstream of repA gene. Plasmid replication is then temperature sensitive and is induced to high levels at 42° C. Moreover, protein synthesis is concomitant with replication, leading to high levels of proteins that are encoded by the vector.
While these runaway-replication plasmid vectors have been used to produce a variety of proteins, including hGCSF and somatotropin, the amount of protein produced has been limited by such factors as the copy number (about 1000 copies per cell), and cell death resulting from runaway replication, thus preventing the use of continuous fermentation techniques. Thus, there is a need for expression vector systems without these limitations.
The present invention discloses genes and gene products from small cryptic plasmids and their use in constructing easily controlled, stable, high copy number replication vectors, and further provides other related advantages.
SUMMARY OF THE INVENTION
Within one aspect of the present invention, controlled-replication plasmid vectors are provided comprising a replication origin region of small cryptic plasmids and a gene encoding the RepA protein. In preferred embodiments the gene encoding RepA is under control of an inducible promoter.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions, and are therefore incorporated by reference in their entirety.
REFERENCES:
patent: 5583040 (1996-12-01), Kaji
Garcia de viedma et al. J. Molecular Biol. 247:211-223, 1995.*
Burian et al., Plasmid 37(1):2-14 (1997).*
Freifelder,Molecular Biology:A Comprehensive Introduction to Prokaryotes and Eukaryotes, Jones and Bartlett Publishers, Inc., Boston, 1983, pp. 797-799.*
George et al., “Current methods in sequence comparison and analysis,” in Macromolecular Sequencing and Synthesis, Selected methods and Applications, 1988, D.H. Schlesinger (ed.), Alan R. Liss, Inc. New York NY, pp. 127-149.*
Boswell et al., “Sequence comparison and alignment: the measurement and interpretation of sequence similarity,” inComputational Molecular Biology:Sources and Methods for Sequence Analysis, 1988, Arthur M. Lesk (ed.), Oxford University Press, New York, NY, pp. 161-178.
Burian Jàn
Kay William W.
Seed IP Law Group
University of Victoria Innovation & Dev. Corp.
Yucel Remy
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