Expression and crystal screening of rice protein GS3 and its complex with Gβ

ObjectiveRegulation mechanism of GS3 protein in rice was investigated, in order to lay foundation for analyzing structure of plant G protein and perfecting regulation network of G protein. MethodN-terminal domain of GS3 protein OSR and C-terminal domain of GS3 protein were expressed by inducing expression vectors pGEX-6p-1 and pET-28b-sumo, respectively. Then the gel filtration columns superdex 200/75 10/300 and ion exchange column Mono Q 5/50 were used to purify protein. The structures of GS3 and co-expression complex of GS3 and Gβ were studied by optimizing crystal and X-ray diffraction. ResultThe constructed recombinant proteins OSR-pGEX-6p-1 and GβN-pET-28b-sumo were co-expressed in Escherichia coli BL21(DE3). Then two bands were obtained by GST beads affinity chromatog-raphy, which were OSR-GST(31 kD) and GβN-sumo(26 kD), respectively. After Ni2+ beads affinity chromatography, two bands were still found. However, SDS-PAGE results showed that, four bands were found, including GST(26 kD), Sumo (17 kD), GβN(9 kD) and OSR(5 kD). The complex of GβN(9 kD) and OSR(5 kD) were obtained by separation and purification with ion exchange column Mono Q 5/50. The purified complex was concentrated to 12 mg/mL for crystal screening, but no crystal was obtained. ConclusionG protein in rice is consistent with the classical heterotrimeric G pro-tein model due to interaction of GS3 and Gβ that GS3 N-terminal domain OSR was integrated with Gβ N-terminal.