Host strain for low uninduced expression of foreign RNA...

Details Host strain for low uninduced expression of foreign RNA... Host strain for low uninduced expression of foreign RNA...

2000-10-12

2003-05-27

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S069100, C435S069200, C435S471000, C435S476000, C435S478000

Reexamination Certificate

active

06569669

ABSTRACT:

BACKGROUND OF THE INVENTION
Expression of proteins toxic to the cell requires that the construct (expression vector) used for final production be made and stored under conditions where the toxic protein is not expressed or is expressed at extremely low levels. It is convenient for this purpose to use expression signals (especially transcriptional promoters) that are not recognized by the cells used during construction (Studier, et al.,
Meth. Enzymol
., 185:60-89 (1990); U.S. Pat. No. 4,952,496 (1990), Studier et al., “Cloning and expression of the gene for bacteriophage T7 RNA polymerase”).
Expression is then accomplished by introducing the expression vector into a special cell line that makes a foreign RNA polymerase that does recognize the expression signal (promoter). Foreign RNA polymerases that recognize highly specific promoter sequences distinct from those recognized by bacteria include those encoded by T7-like phages and may be selected from the group consisting of
Escherichia coli
phages T3, .phi.I, .phi.II, W31, H, Y, A1122,cro, C21, C22 and C23; Pseudomonas putida phage gh-1
; Salmonella typhimurium
phage Sp6
; Serratia marcescens
phage IV; Citrobacter phage VIIII; and Klebsiella phage number 11. The RNA polymerase of T3 has been used-this way (Morris, et al.,
Gene
, 41:193-200 (1986)). In principle, such foreign RNA polymerases could include those of Archaea or Eukaryotes. Such specialized bacteriophage RNA polymerases have also been used in eukaryotes as well (U.S. Pat. No. 5,550,035, Moss et al, “Prokaryotic expression in eukaryotic cells” (1996)).
Further, it is convenient to provide for regulation of the expression and activity of this foreign RNA polymerase, (Dubendorff and Studier,
J Mol Biol
, 219:45-59 (1991)). This is done in common cases in two ways: by providing a binding site (operator) for a transcriptional repressor as part of the expression signals for the foreign RNA polymerase gene itself; and by providing additional operators as part of the expression signals for the gene of interest. In common examples and in the present invention, the foreign RNA polymerase used is that of bacteriophage T7, the transcriptional regulator used is the LacI repressor, and the operator is the lac operator.
Accordingly, commonly used strains, e.g. BL21(DE3) and ER2566, provide a chromosomal copy of the lacI gene to facilitate negative regulation of expression of the T7 RNA polymerase before introduction of the plasmid vector carrying the gene of interest. In addition, to provide sufficient LacI to also repress copies of the T7 promoter carried on the plasmid vector itself, many such expression vectors provide further copies of the lacI gene (e.g. (Maneewannakul, et al.,
Plasmid
, 31:300-7 (1994), Munson, et al.,
Gene
, 144:59-62 (1994), Andrews, et al.,
Gene
, 182:101-9 (1996)).
SUMMARY OF THE INVENTION
The invention relates to regulated expression of toxic genes by cells that use a foreign RNA polymerase to transcribe the gene for the toxic product. Specifically, the invention relates to provision of high levels of a negative. regulator of the expression of the foreign RNA polymerase or of the toxic gene or both, which high level of negative regulator is established in the cell before the introduction of the toxic gene.
In one preferred embodiment, the present invention provides an expression strain carrying a copy of the T7 RNA polyrderase gene together with a copy of the wild type lacI gene similar to other such strains available, but with the additional provision of further copies of the lacI gene with a mutated promoter such that 10-fold higher levels of the repressor protein are expressed than in the standard host.


REFERENCES:
patent: 5830694 (1998-11-01), Studier et al.
patent: 6165749 (2000-12-01), Sagawa et al.
Studier, et al., Meth. Enzymol., 185:60-69 (1990).
Morris, et al., Gene 41:193-200 (1986).
Dubendorff and Studier, J. Mol. Biol. 219:45-59 (1991).
Maneewannakul, et al., Plasmid 31:300-307 (1994).
Munson, et al., Gene 144:59-62 (1994).
Andrews, et al., Gene 182:101-109 (1996).
Gilbert and Müller-Hill, Proc. Natl. Acad. Sci. USA 56:1891-1898 (1996).
Kennell and Riezman, J. Mol. Biol. 114:1-21 (1977).
Ausubel, et al., Current Protocols in Molecular Biology, John Wiley and Sons pp. 1.5-1.15, 13.4-13.6 and 9.9-9.14 (1999).
Yanisch-Perron, et al., Gene, 33:103-119 (1985).
Calos, Nature, 274:762-765 (1978).
Müller-Hill, et al., Proc. Natl. Acad. Sci. USA., 59:1259 (1968).
Hawley and McClure, Nucleic Acids Res. 11:2237-2255 (1983).
Angrand, et al., Nucleic Acids Res. 27:e16 (1999).
Payne, et al. J. Hum. Hypertens, 13:845-848 (1999).
Posfai, et al., J. Bacteriol. 179:4426-4428 :(1997).
Andrew et al. A tightly regulated high level expression vector that utilizes a thermosensitive lac repressor: production of the human T Cell receptor VB5.3 inEscherichia coliGene 182 1996 101-109.*
Munson et al. ColE1-compatible vectors for high-leveln expression of cloned DNAs from the T7 promoter 1994 Elsevier Science B. V. 0378-1119.*
Szafranski et al. A new approach for containment og microorganisms: Dual control of streptavidin expression by antisense RNA and the T7 transcription system Pro Natl acad Scri USA vol. 94 pp. 1059-1063 Feb. 1997.*
Studier et al. Use of T7 RNA Polymerase to Direct Expression of Cloned Genes Methods in Enzymolgy vol. 185.

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