A reconstruction of the gene for ribulose bisphosphate carboxylase from Rhodospirillum rubrum that expresses the authentic enzyme in Escherichia coli

Abstract Escherichia coli plasmid pRR36, which expresses Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) as a fusion protein [Nargang et al., Mol. Gen. Genet. 193 (1984) 220–224], was used to construct a new clone of the carboxylase gene ( rbc ) whose expression product is the wild-type enzyme. This construction entailed removing all lacZ -coding sequences and a portion of the 5'-noncoding leader of the R. rubrum rbc gene. The highest specific activity of carboxylase was observed with an expression vector which juxtaposed the trp-lac (tac) hybrid promoter with the R. rubrum ribosome binding site and the rbc structural gene. The carboxylase expressed in E. coli JM107 was purified to near homogeneity and, based on subunit M r and specific enzymic activity, the isolated protein appeared indistinguishable from authentic ribulose bisphosphate carboxylase from R. rubrum . N-terminal sequence analyses of the cloned enzyme verified that the cloned and wild-type enzymes are the same.