Amplification of the UvrA Gene Product of Escherichia Coli to 7% of Cellular Protein by Linkage to the PL Promoter of PKC30

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1982 Mar 1

PMID 7043463

Citations 7

Authors

G H Yoakum

A T Yeung

W B Mattes

L Grossman

Affiliations

Soon will be listed here.

Abstract

We have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under pL control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, we developed a genetic selection using the basal level of expression (30 degrees C) from pL in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products. In addition, a post-UV-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under pL control rapidly. This should prove generally useful for isolating genes inserted at the Hpa I site of plasmid pKC30 with the following characteristics: (i) genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and (ii) production of high-level amplification for the gene product of interest by screening for post-UV-irradiation temperature inducibility of polypeptides synthesized from hybrid plasmids. The level of amplification obtained for the uvrA gene product from pGHY5003 is approximately 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase Escherichia coli.

Citing Articles

Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters.

Valdez-Cruz N, Caspeta L, Perez N, Ramirez O, Trujillo-Roldan M Microb Cell Fact. 2010; 9:18.

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Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli.

Walker G Microbiol Rev. 1984; 48(1):60-93.

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High-level production of biologically active human alpha 1-antitrypsin in Escherichia coli.

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Enzymatic properties of purified Escherichia coli uvrABC proteins.

Yeung A, Mattes W, Oh E, Grossman L Proc Natl Acad Sci U S A. 1983; 80(20):6157-61.

PMID: 6312446 PMC: 390162. DOI: 10.1073/pnas.80.20.6157.

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