Immunoblot Analysis of the Expression of Genes for Barly Rubisco Activase in E. coli
1998
Roh Kwang-Soo | Kwon Mo-Sun Department of Biology, Keimyung University, Taegu 704-701 (Korea Republic)
Rubisco activity during photosynthesis is regulated by the rubisco activase, which facilitates the dissociation of RuBP and other inhibitory sugar phosphates from the active site of rubisco in an ATP-dependent reaction. In this paper, barley Rca genes (RcaA1, RcaA2 and RcaB) were expressed in E. coli and the activity of rubisco activase expressed was assayed biochemically by chromatography. Then the protein was identified electrophoretically by SDS-PAGE and detected immunologically by Western blot analysis using polyclonal antibodies raised against the kidney bean rubisco activase as probe. The band pattern of purified proteins on the polyacrylamide gel showed two polypeptides of 46 kD and 42 kD. Anti-rubisco activase antibodies reacted specifically with both polypeptides of 46 kD and 42 kD present in the crude extracts of E. coli transformants. Therefore, it was found that the genes of barley rubisco activase was successfully expressed in E. coli as active forms of 46 kD and 42 kD.
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