Tetracycline promoter for the stringently regulated production o

Chemistry: molecular biology and microbiology – Vector – per se

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435 697, 4352523, 536 234, 536 241, C12N 1563, C12N 1564, C12N 1570

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058495762

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BRIEF SUMMARY
SUMMARY OF THE INVENTION

The present application is the national stage entry of PCT/EP95/01862 filed on May 17, 1995.
The present invention concerns a prokaryotic vector which contains a regulatable expression control sequence which can be repressed by the repressor of the tetracycline resistance gene, a prokaryotic cell transformed with this vector and the use of the vector or the cell in a process for the production of polypeptides in prokaryotes by genetic engineering.
Inducible promoter systems have proven to be very suitable for the production of heterologous proteins in E. coli since synthesis of the recombinant gene product often has a toxic effect on the bacterial cell which impairs its growth and viability. In particular when the foreign protein is secreted into the periplasmatic space of the host cell, it is advisable to ensure a strict repression of the promoter not only in the course of the procedures for producing the vector but also in order to achieve high cell densities with the transformed bacteria before the actual protein production. The bacterial secretion of antibody fragments is a typical example in this respect since, due to the heterologous gene expression, a toxic and lytic effect is observed on the bacterial cell (Plukthun and Skerra, Methods Enzymol. 178 (1989), 497-515).
An inducible promoter system that is used particularly frequently is the lac promoter and its derivatives that can be induced by isopropyl-.beta.-D-thiogalactopyranoside (IPTG) e.g. the mutant lacUV5 or the tac fusion promoter (Reznikoff and Gold (1986), in: Maximizing gene expression, Butterworth Publishers, Stoneham, Mass.). The use of the lac promoter to produce antibody fragments in bacteria by genetic engineering is described for example by Skerra (dissertation, LMU Munich, Faculty for Chemistry and Pharmacy, 1983).
However, the strength of the transcription by the lac promoter is coupled to the genotype and metabolism of the host cell via the endogenous concentration of lac repressor molecules on the one hand and via the catabolite repression effect on the other hand. Thus when using a lac expression system considerable variations in the expression level are observed with a given vector depending on the host strain used. This may be either due to a reduced inducibility--particularly if the lac repressor is coded chromosomally as well as by a plasmid and thus is present in excessive amounts--or to a dying of the cells or a loss of plasmid before the induction as a result of inadequate repression.
In addition to the lac promoter other regulatable promoter systems have also been used, but, most of them have considerable disadvantages especially if a moderate secretion of the foreign gene product or an expression at a reduced temperature is desired in order to favour the protein folding process. Therefore there is a great need for the provision of a prokaryotic expression control sequence which is largely decoupled from the individual properties of the bacterial host cell and can be reversibly induced in a simple and cost-effective manner.
Plasmid vectors are described in a publication by De la Torre et al. (Plasmid 12 (1984), 103-110) in which the gene expression is partially regulated by tetracycline. These vectors contain the regulatory region of the tetracycline resistance gene from the transposon Tn10. This region originally causes the expression of the tetracycline resistance gene in one direction and the expression of the tetracycline repressor structural gene in the other (Bertrand et al., Gene 23 (1983), 149-156). In an interaction with the operator the tetracycline repressor protein inhibits the expression in both directions and is thus subject to autoregulation. The repression of a foreign gene instead of the tetracycline resistance gene under the control of this tetracycline promoter is achieved by co-transformation with a compatible plasmid which contains the tetracycline repressor gene which is likewise under the control of the tetracycline promoter. In host cells containing the two plasmids an

REFERENCES:
patent: 5464758 (1995-11-01), Gossen et al.
Bertrand et al. Construction of a single-copy promoter vector and its use analysis of regulation of the transposon Tn10 tetracycline resistance determinant. Journal of Bacteriology. vol. 158, No. 3, pp. 910-919, Jun. 1984.
Gatz et al. Regulation of a modified CaMV 35S promoter by the Tn10-encoded Tet repressor in transgenic tobacco. Molecular and General Genetics. vol. 227, pp. 229-237, 1991.
Chopra et al. Sensitive biological detection method for tetracyclines using a tetA-lac7 fusion system. Antimicrobial Agents and Chemotheraphy. vol. 34, No. 1, pp. 111-116, Jan. 1990.
Wray et al., Journal of Bacteriology, "Identification of Repressor Binding Sites Controlling . . . ", vol. 156, Dec. 1993, No. 3.
Schiweck et al, Proteins, "Fermenter Production of an Artificial Fab Fragement, Rationally . . . ", 23:561-565 (1995).
Skerra, Gene, "Use of the tetracycline promoter for the tightly regulated production of a murine . . . ", 151 (1994) 131-135.

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