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.
Keywords: Ribulose 1,5-bisphosphate carboxylase/oxygenase, Protein purification, Chlamydomonas reinhardtii, Rubisco, Density gradient centrifugation
Materials and Reagents
Equipment
Procedure
Representative data
The final preparation should contain more than 95% of Rubisco protein being substantially free of nucleic acids (A280/A260 ratio higher than 1.9). Rubisco concentration may be calculated from the UV absorbance assuming an extinction coefficient of ε1% = 15.9 dl·g-1·cm-1. at 280 nm. Typical final concentration of Rubisco is about 1 mg/ml. Figure 3 illustrates the degree of purity as ascertained by Coomassie Blue-stained SDS-PAGE. The described protocol leads to high purity Rubisco that might be used even for crystallization of the enzyme. In case that such a degree of purity is not needed, the protocol might be shortened omitting the anion exchange chromatography (section C). After the sucrose gradient centrifugation, Rubisco is already more than 80% of the total protein in the extract (see Figure 3B), although there is still a significant contamination of nucleic acids. Figure 3. Coomassie-stained SDS-PAGE analysis of Rubisco purification from C. reinhardtii. Arrows indicate the position of the large (LS) and small (SS) subunits of Rubisco on a 14% acrylamide SDS-PAGE gel. A. Final preparation of purified Rubisco (0.25 μg, lane P) run in parallel to markers (MM) of molecular mass (in kDa). B. Fractions (0.7 ml each) collected from the bottom of the sucrose gradient after centrifugation. The red line above indicates the fractions to be pooled for further purification by anion exchange chromatography.
Notes
Recipes
Acknowledgments
This work was supported by a grant (UV-INV-AE14-269247) of the University of Valencia. The protocol is a detailed and expanded version of the one published as supplementary information in Sudhani and Moreno (2015).
References
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