The cloning and expression in Escherichia coli of RuBP carboxylase/oxygenase large subunit genes

This chapter describes the cloning and expression in Escherichia coli of RuBP carboxylase/oxygenase (RuBisCo) large subunit genes. The application of recombinant DNA methodologies to the analysis of RuBisCo structure and function is directed toward the resolution of problems requiring that large quantities of the protein product of cloned RuBisCo genes be accessible for enzymological analysis. The production of an active enzyme in E. coli involves the construction of an expression vector that permits coordinate transcription and translation of two genes. Because, in photosynthetic eukaryotes, one or both subunits are translated as precursors, the genes from these organisms must be restructured to ensure that the primary translation product in E. coli is functionally equivalent to that found in the mature form of the enzyme. The RuBisCo expression vector based on the R. rubrum gene is suitable for most investigations––such as RuBisCo reaction mechanism––the weak homology among this gene, and the large subunit genes from most other organisms effectively forbids the exploitation of natural variation in the primary structure by such approaches as the formation of synthetic hybrid genes.