Purification of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunits suitable for reconstitution and assembly of active L8S8 enzyme

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans was reconstituted in vitro from extracts of Escherichia coli strains that separately express large and small subunits. This reconstitution system was shown to be useful for monitoring the appearance of dissociated or fractionated subunit preparations. Recombinant large subunits were purified to a state of homogeneity and retained reconstitution capacity in the presence of added small subunits. The purified large subunits appeared to be in the form of an octamer, probably an L8 structure, and showed 0.15% of the carboxylase activity of the purified L8S8 enzyme. Purified large subunit octamers are disrupted by nondenaturing PAGE; however, the octamer is stable to electrophoresis in the presence of exogenous protein.