Expression,purification and activity identification of SCR15-18 domain of human complement receptor 1 in Pichia Pastoris / 第三军医大学学报

ABSTRACT

Objective To express the short consensus repeat 15-18 (SCR15-18) domain of human complement receptor 1 (CR1) in Pichia pastoris as a secreted protein in order to found a base for large-scale industry fermentation. Methods The gene fragment of CR1-SCR15-18 was amplified by PCR from plasmid pET32a-sCR1-SCR15-18. The obtained sequence was subcloned into Pichia pastoris secretory expression vector pPIC9. After identification the recombinant plasmid pPIC9-CR1-SCR15-18 was electrotransported into the yeast. Positive clones were identified using colony PCR. Positive recombinants were fermented in shake flaskes and induced with methanol. The recombinant proteins were identified with SDS-PAGE and Western blot analysis. After the recombinant protein was purified by Ni-NTA agarose metal chelate affinity chromatography, inhibiting complement hemolysis testing was used to detect the biological activity. Results Recombinant expression plasmid pPIC9-CR1-SCR15-18 was successfully constructed. SDS-PAGE and Western blot analysis revealed that the target gene was successfully expressed in the yeast and the recombinant protein was successful secreted in the culture supernatant. After purification, the protein inhibited complement hemolysis in vitro. Conclusion CR1-SCR15-18 is successful expressed in Pichia Pastoris with high activity of inhibiting complement hemolysis.