Molecular Cloning，Prokaryotic Expression and Purification of Sheep Sperm Equatorial Segment Protein 1(SPESP1)

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.023

Abstract

Abstract:

The purpose of this study is to clone sheep sperm equatorial segment protein 1 cDNA(SPESP1), express SPESP1 in prokaryotic cells. Firstly, SPESP1 cDNA was amplified with total cDNA of sheep testicular as template and the designed primers according to the sequence of sheep genome in GenBank. Then the amplified SPESP1 cDNA was cloned into the expression vector pGEX-4T1 to construct pGEX-SPESP1, which was transferred into Escherichia coli BL21 for expression and the conditions of expression were optimized. The purified fusion protein by SDS-PAGE gel-slicing was identified by Western blotting. By sequence alignment, two base pairs of cDNA sequence were different between the cloned SPESP1 and the predicted one in GenBank, which led to 1 amino acid varied. The expression of the recombinant fusion protein in E. coli BL21 succeeded under the optimal condition of 37℃ and 0.005% of IPTG for 4 h. SDS-PAGE and Western blotting analysis revealed that a protein at band of approximate 64 kD could be recognized by the antibody of anti-GST and anti-sheep-SPESP1, which was in accord with the prediction, and this implied that expression of fusion protein was achieved. In conclusion, successful gain of purified fusion protein GST-SPESP1 lays a foundation for the functional studies of SPESP1 in the membrane fusion of sperm and egg.

Key words: sheep, SPESP1, cDNA cloning, prokaryotic expression, protein purification

Ma Yuanyuan, Cheng Feiyue, Xing Wanjin. Molecular Cloning，Prokaryotic Expression and Purification of Sheep Sperm Equatorial Segment Protein 1(SPESP1)[J]. Biotechnology Bulletin, 2015, 31(7): 155-160.

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