Amplification of the uvrA gene product of Escherichia coli to 7% of cellular protein by linkage to the pL promoter of pKC30.

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Abstract
A hybrid plasmid pKC30-uvrA plasmid (pGHY5003) was constructed in which transcription of the E. coli uvrA gene can be induced under pL control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, a genetic selection was done using the basal level of expression (30.degree. 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: genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and 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 amplication obtained for the uvrA gene product from pGHY5003 is .apprx. 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase. E. coli.