Cloning and expression of the Rhodospirillum rubrum ribulosebisphosphate carboxylase gene in E. coli

Summary

A genomic library of Rhodospirillum rubrum DNA was constructed in the phage replacement vector λ1059. Recombinant phage carrying the gene for ribulose 1,5-bisphosphate (RuBP) carboxylase were identified by radioimmune blotting of plaques. A 6.6 kb EcoRI fragment from one of the immunologically positive phage was subcloned into pBR325 and a plasmid which conferred low levels of enzyme activity in E. coli was recovered. Expression of the gene was dependent upon the orientation of the restriction fragment in pBR325, suggesting that transcription originated on a plasmid promoter. In order to increase expression, a new plasmid was constructed by replacing the tetracycline resistance determinant of pBR322 with a restriction fragment from phage M13mp7 which encoded part of the lac operon. A 2.4 kd restriction fragment containing the RuBP carboxylase gene was then cloned into the unique BamHI site of the lac DNA. Induction of lac-transcription resulted in a high level of expression of a fully functional RuBP carboxylase enzyme, which was purified to homogeneity. Southern hybridization analysis of the R. rubrum genome with a restriction fragment encoding part of the enzyme indicated only one copy of the gene.