Prokaryotic Expression and Purification of P53 Protein

Objective To construct a prokaryotic expression vector for P53 gene, express fusion protein in E.coli and purify the expressed product.Methods The full-length of P53 gene was amplified by PCR using plasmid pBabe-P53R as a template and cloned into vector pGEX-4T-1.The constructed recombinant plasmid pGEX-4T-1-P53 was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was re-naturalized by detergents with various formula and purified by Glutathione Sepharose 4B gel affinity chromatography.Results Restriction analysis proved that recombinant plasmid pGEX-4T-1-P53 was constructed correctly.The relative molecular mass of expressed GST-P53 fusion protein was about 80 000.The optimal time for induction with IPTG was 1 h.The optimal detergent for re-naturalization of the fusion protein consisted of 1% Triton X-100 and 1 mmol / L EDTA.The purified fusion protein showed specific reactions with goat anti-human GST and mouse anthuman P53 anti-bodies.One milligram of purified protein was obtained from 500 ml of bacterial liquid.Conclusion The prokaryotic expression vector for P53 gene was successfully constructed, and GST-P53 fusion protein was expressed and purified, which laid a foundation of study on relationship between Np95-like ring finger protein(NIRF)and P53 gene.