Prokaryotic expression,purification and identification of human P311 protein

Objective To express, purify and identify human P311 protein, a candidate protein in hypertrophic scar formation. Methods The cDNA sequence of human P311 obtained by PCR was cloned into the prokaryotic expression vector pGEX-4T-1 containing glutathione-s-transferase (GST). pGEX-4T-P311 was transformed into E. coli BL21(DE3). The expression of GST-P311 fusion protein was induced by IPTG, identified by both SDS-PAGE and Western blotting, and then purified with glutathione-sepharose beads. Results The restriction endonuclease digestion and the sequencing of recombinant plasmid demonstrated that pGEX-4T-P311 vector was successfully constructed. The relative molecular weight of expressed protein was about 34×103 which was as the same as the objective protein, and the corresponding protein was identified by the immunoblotting with antibody against GST. Conclusion pGEX-4T-P311 vector is correctly constructed, and the GST-P311 fusion protein is successfully expressed in E. coli BL21(DE3)and purified.