Preliminary Study For Expression Of Recombinant Mouse Sperm-specific Catsper1_S1-S6 In Pichia Pastoris

AIM CatSperl is a putative sperm cation channel which is specifically located in the principal piece of the sperm tail and expressed in spermary. Its structure resembles a single, six-transmembrane-spanning repeat of the voltage-dependent Ca²⁺ channel four repeat structure. Targeted disruption of this gene results in remarkable decrease of sperm motility and male sterility. CatSperl represents an excellent target for contraceptives. In this study, we constructed a recombinant plasmid pPICZaA-Catsperl_S1-S6-and built an expression and purification system in order to obtain the active 6his-tag fused Catsperl_S1-S6 protein for further study. METHODS The total RNA extracted from mouse testis was used as template to amplify the cDNA encoding the transmembrane domain S1-S6 by RT-PCR. After the gene fragments of S1-S6 and pPICZαA were digested by EcoR I and Sal I, these two digested fragments were connected in common ways and transformed into the E.coli DH5α competent cells. The positive clones were screened with Zeocin and identified by PCR. Then, the combinational plasmid was analysed by restriction endonucleases and sequcecing analysis. The correct combinational plasmid was linealized by Sac I and transformed into Pichia pastoris GS115 strains which was screened with Zeocin.The recombinant Pichia pastoris strains were identified by PCR, and then the positive clone was cultured following the manufacturer's protocol to express the Catsperl_S1-S6 protein whose expression was induced by methanoLAt last, SDS-PAGE and Western blotting were used to analyses the aim protein that was purified with Ni-chelating affinity chromatography column..RESULES Sequcecing analysis showed that the RT-PCR product was our aim gene. The combinational plasmid was also tested to be correct by restriction endonucleases and sequcecing analysis.SDS-PAGE and Western blotting analysis showed that the apparent molecular weight of the expressed Catsperl_S1-S6 proteins in yeast was about 43kD.CONCLUSION The recombinant plasmid pPICZaA-Catsperl_S1-S6 was successfully constructed. An expression strain of GS115-Catsperl_S1-S6 was obtained. The recombinant protein Catsperl_S1-S6 could be expressed by yeast in secreted form.