Expression of Cyanobacterial and Higher-Plant Ribulose 1,5-Bisphosphate Carboxylase Genes in Escherichia coli

Expression strategies for the synthesis of higher-plant and cyanobacterial RuBP carboxylase genes in Escherichia coli have been developed to facilitate the study of the assembly pathway and properties of the enzyme's large (L) and small (S) subunit proteins. The genes for the L and S subunits of the RuBP carboxylase of wheat and of a cyanobacterium, Synechococcus 6301 have been cloned into bacteriophage and plasmid vectors such that they are transcribed and translated in E. coli. To data no RuBP carboxylase activity has been detected in extracts prepared from E. coli cells synthesizing the wheat L and S subunits, although both gene products were present and soluble. Sucrose gradient analysis of cell extracts from E. coli synthesizing both L and S demonstrated that the soluble wheat L polypeptide was present as a large protein aggregate that contained no S subunits. With the cloned cyanobacterial genes, RuBP carboxylase activity could be recovered in E. coli cell extracts when the L and S gene products were synthesized from genes present on the same, or separate, replicons. Solubility and sedimentation studies of the cyanobacterial L subunits synthesized in the absence of S showed that the L subunit was soluble and present in E. coli as an L$_8$ structure. The E. coli extracts containing only the L subunit exhibited no detectable RuBP carboxylase activity. Infection of the E. coli cells containing L subunits with an M13 phage expressing the cyanobacterial S gene led to the assembly of functional RuBP carboxylase in these cells. This demonstrates the essential role of the S subunit in allowing the formation of an active enzyme.