Prokaryotic expression and purification of Spink12,a mouse epididymal secretary protein / 解放军医学杂志

ABSTRACT

Objective To study the effect of immunocontraception of Spink12,the fusion protein 6?His-Spink12 was induced to express in E.coli BL21,and purified.MethodsThe total mouse epididymal RNA was extracted,and the Spink 12 protein coding exon without signal peptide sequence was amplified by RT-PCR,digested by restrictive enzyme Nde Ⅰ and EcoR Ⅰ,and then cloned into the prokaryotic expression vector pET28 (a) which contained the 6?His tag in N terminal.The recombinant plasmid pET28a-Spink12,after being confirmed by enzyme digestion and sequencing,was transformed into the competent cells of E.coli BL21.The fusion protein 6?His-Spink12 was expressed by IPTG induction and analyzed by SDS-PAGE and Western blotting.The products were purified by Ni-NTA under native condition with different imidazole gradients and characterized by Western blotting with anti-His monoclonal antibody.ResultsThe results of digestion and sequencing indicated that the recombinant prokaryotic expression vector pET28a-Spink12 was successfully constructed.After E.coli BL21 was transformed with pET28a-Spink12 and induced with IPTG,the recombinant target protein of about 9 kDa was obtained by SDS-PAGE analysis,which was coincident with the theoretical value.The fusion protein was of 23.5% of the total E.coli cell proteins.The Western blotting analysis with anti-His monoclonal antibody showed that the band was fusion protein 6?His-Spink12.After purified with Ni-NTA,a higher quality of fusion protein 6?His-Spink12 was obtained in a purity of 91.1%.ConclusionsThe fusion protein 6?His-Spink12 has been successfully expressed in E.coli BL21 and purified.It may provide a base for the study of the effect of Spink12 on immunocontraception.