Construction of plasmids for expression of Rous sarcoma virus transforming protein, p60src, in Escherichia coli

Abstract

We have constructed plasmids that direct the synthesis of the Rous sarcoma virus transforming gene (src) product (p60src) in Escherichia coli. A 203-base-pair lac promoter-operator DNA encoding the first eight amino acids of beta-galactosidase was ligated to the 5' end of the src gene from the Prague A strain of Rous sarcoma virus (PrA-RSV) which had been cloned in pBR325. Antiserum, from a tumor-bearing rabbit, directed against pp60src was used to screen bacteria containing the recombinant plasmid for a protein of approximately 60,000 daltons, and several colonies producing a protein immunologically related to pp60src were detected. Partial proteolytic cleavage analysis revealed that the src-related protein produced in bacteria is structurally similar to pp60src immunoprecipitated from PrA-RSV-infected chicken cells. Partially purified src protein from E. coli can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. Tryptic phosphopeptide analysis demonstrated that the catalytic subunit phosphorylated a serine-containing tryptic peptide in the bacterial src protein that comigrated with the phosphoserine-containing tryptic peptide of pp60src immunoprecipitated from 32P-labeled PrA-RSV-infected chicken cells.