Soluble expression of Spinach psbC gene in Escherichia coli and in vitro reconstitution of CP43 coupled with chlorophyll a only

Abstract

CP43 is a chlorophyll a (Chl a) and β-carotene (β-Car) binding protein encoded by psbC gene. In this study, psbC gene isolated from Spinach was expressed in Escherichia coli in soluble state. After lysis of the cells, the apoproteins purified by nickel affinity chromatography were examined by SDS-PAGE and Western-blot. Next, reconstitution experiment was carried out in vitro and the formation of stable pigment-protein complex was analyzed by partially denaturing electrophoresis. After purifying reconstituted CP43 (rCP43) from free pigments (FPs) by sucrose gradient ultracentrifugation and subsequently ion exchange chromatography (IEC), the eluate was analyzed by partially denaturing electrophoresis to confirm stability of the reconstructed complex. Finally, analyses of spectroscopic character of the eluate revealed that in vitro reconstitution was achieved and FPs were completely removed from the pigment-protein complex. Comparison between the absorption spectra of the rCP43 and native CP43 (nCP43) showed the lack of peaks between 450 and 500 nm, illustrating that the β-Car was stripped off rCP43. In brief, it is feasible to obtain a reconstituted protein binding Chl a only, indicating that the occupancy of the β-Car site has small impact on the stabilization of CP43. However, β-Car shows strong interaction with Chl a, inducing the hyperchromic effect in blue region of spectrum and the blue shift of the 438.5 nm and 673.5 nm absorption band to 437 nm and 671 nm respectively. To some extent, our research is suggestive that β-Car, coupled loosely with CP43, contributes to the precise orientation of Chl a in vivo.

Keywords: Partially denaturing polyacrylamide green gel electrophoresis; Photosystem II; Reconstituted CP43 binding only chlorophyll a; Reconstitution in vitro; Soluble expression; Visible absorption and fluorescence spectrometry.