Purification of Rubisco from Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii is a model organism for chloroplast studies. Besides other convenient features, the feasibility of chloroplast genome transformation distinguishes this unicellular alga as ideal for the manipulation of chloroplastic gene expression aiming biotechnological goals, such as improved biofuel and biomass production. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) is the photosynthetic carbon-fixing enzyme which is considered crucial for biomass accumulation in algal cultures. Purification of wild type and site-directed mutants of Rubisco in C. reinhardtii is usually performed to study its catalytic properties and assess the carbon-fixing potential of the strains. In this protocol Rubisco is extracted through sonication of cell pellets, and purified by ammonium sulfate precipitation, sucrose gradient centrifugation (Goldwaithe and Bogorad, 1975) and anion exchange chromatography.