Expression of sarco/endoplasmic reticulum Ca(2+)-ATPase from Bombyx mori by baculovirus expression system.
- Author:
Xin WANG
;
Yi LI
;
Huifang CHEN
;
Xiaoying ZHOU
;
Kang XIE
;
Ping ZHAO
- Publication Type:Journal Article
- MeSH:
Animals;
Baculoviridae;
Bombyx;
enzymology;
Cell Line;
Genetic Vectors;
Recombinant Proteins;
biosynthesis;
Sarcoplasmic Reticulum Calcium-Transporting ATPases;
biosynthesis;
Transfection
- From:
Chinese Journal of Biotechnology
2015;31(3):421-430
- CountryChina
- Language:Chinese
-
Abstract:
Sarco/endoplasmic reticulum Ca(2+)-ATPase (Serca) is responsible for transporting Ca2+ into the endoplasmic reticulum and maintaining a suitable calcium environment in cells. The suitable calcium environment created by BmSerca is vital for the growth and development of silkworm. With a large molecular weight and 10 transmembrane domains, Serca is very difficult to express in Escherichia coli expression system. In order to obtain recombinant Serca with biological activity, pFastBac Dual vector was used to construct a binary baculovirus expression vector for expressing egfp and serca in cells. After transfection and infection, EGFP and Serca were expressed successfully in BmN-SWU1 cell line. Fluorescent observation revealed that the expression patterns of EGFP and Serca in infected cells were the same. Western blotting analysis showed that the recombinant proteins were about to express in cells 48 h post infection and highly expressed 96 h post infection. Ca(2+)-ATPase activities assays were used to evaluate the enzyme activities of recombinant Serca and found that the enzyme activities increased significantly after infection. The obtained data showed that this binary baculovirus expression system can be successfully used to express Serca with biological activity. The expression of Serca protein with this system is useful for further research on the function of Serca.
