Abstract
Salt-induced overexpression of genes cloned downstream of the phage T7 phi10 promoter was demonstrated in an Escherichia coli strain (GJ1158) which carries a single chromosomally integrated copy of the gene for phage T7 RNA polymerase under transcriptional control of the cis-regulatory elements of the osmoresponsive proU operon. Plasmids that have been constructed to obtain overproduction of individual target gene products in strain BL21(DE3) (by addition of isopropyl-beta-D-thiogalactopyranoside as an inducer) can directly be transformed into GJ1158. The NaCl induction regimen was also shown to be associated with a decreased propensity for sequestration of overexpressed target proteins within insoluble inclusion bodies.
Full Text
The Full Text of this article is available as a PDF (629.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Blackwell J. R., Horgan R. A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett. 1991 Dec 16;295(1-3):10–12. doi: 10.1016/0014-5793(91)81372-f. [DOI] [PubMed] [Google Scholar]
- Csonka L. N. Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev. 1989 Mar;53(1):121–147. doi: 10.1128/mr.53.1.121-147.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dattananda C. S., Rajkumari K., Gowrishankar J. Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene. J Bacteriol. 1991 Dec;173(23):7481–7490. doi: 10.1128/jb.173.23.7481-7490.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldberg A. L. The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur J Biochem. 1992 Jan 15;203(1-2):9–23. doi: 10.1111/j.1432-1033.1992.tb19822.x. [DOI] [PubMed] [Google Scholar]
- Gowrishankar J. Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation. J Bacteriol. 1985 Oct;164(1):434–445. doi: 10.1128/jb.164.1.434-445.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gowrishankar J., Manna D. How is osmotic regulation of transcription of the Escherichia coli proU operon achieved? A review and a model. Genetica. 1996 May;97(3):363–378. doi: 10.1007/BF00055322. [DOI] [PubMed] [Google Scholar]
- Gutierrez C., Devedjian J. C. Osmotic induction of gene osmC expression in Escherichia coli K12. J Mol Biol. 1991 Aug 20;220(4):959–973. doi: 10.1016/0022-2836(91)90366-e. [DOI] [PubMed] [Google Scholar]
- Herbst B., Kneip S., Bremer E. pOSEX: vectors for osmotically controlled and finely tuned gene expression in Escherichia coli. Gene. 1994 Dec 30;151(1-2):137–142. doi: 10.1016/0378-1119(94)90644-0. [DOI] [PubMed] [Google Scholar]
- Igarashi K., Ishihama A. Bipartite functional map of the E. coli RNA polymerase alpha subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell. 1991 Jun 14;65(6):1015–1022. doi: 10.1016/0092-8674(91)90553-b. [DOI] [PubMed] [Google Scholar]
- Kishore U., Chauhan M., Choudhury N. R., Khatri G. S. Overexpression of Escherichia coli single stranded DNA binding protein under bacteriophage T 7 promoter. Indian J Biochem Biophys. 1994 Aug;31(4):249–253. [PubMed] [Google Scholar]
- Makrides S. C. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev. 1996 Sep;60(3):512–538. doi: 10.1128/mr.60.3.512-538.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nagata S., Adachi K., Shirai K., Sano H. 23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli. Microbiology. 1995 Mar;141(Pt 3):729–736. doi: 10.1099/13500872-141-3-729. [DOI] [PubMed] [Google Scholar]
- Radha V., Kamatkar S., Swarup G. Binding of a protein-tyrosine phosphatase to DNA through its carboxy-terminal noncatalytic domain. Biochemistry. 1993 Mar 9;32(9):2194–2201. doi: 10.1021/bi00060a010. [DOI] [PubMed] [Google Scholar]
- Rajkumari K., Kusano S., Ishihama A., Mizuno T., Gowrishankar J. Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli. J Bacteriol. 1996 Jul;178(14):4176–4181. doi: 10.1128/jb.178.14.4176-4181.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
- Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
- Tabor S., Huber H. E., Richardson C. C. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem. 1987 Nov 25;262(33):16212–16223. [PubMed] [Google Scholar]
- Vidal-Ingigliardi D., Raibaud O. A convenient technique to compare the efficiency of promoters in Escherichia coli. Nucleic Acids Res. 1985 Aug 26;13(16):5919–5926. doi: 10.1093/nar/13.16.5919. [DOI] [PMC free article] [PubMed] [Google Scholar]