Prokaryotic expression and purification of His-tagged-STAT4 (565-748 amino acid) fusion protein / 中国免疫学杂志

ABSTRACT

Objective:To construct His-tagged peptide of human STAT4 (565-748 amino acid) expression vector and induce its expression in Escherichia coli,followed by purification.Methods:STAT4 gene fragment encoding C-terminal peptide of 565-748 amino acid was amplified by PCR using pEGFP-STAT4 as the template.The PCR product was inserted into prokaryotic expression vector pET-28a and was transformed into component E.coli BL21 cells.By isopropyl-β-D-thiogalactoside(IPTG) induction,fusion protein was found to be expressed in the inclusion body and was denatured by using the urea denaturation buffer followed by renaturation and purifi-cation.Finally the purified protein was confirmed by Western blot.Results:The STAT4 truncated gene encoding 565-748 amino acids peptide was amplified by PCR and inserted into pET-28a vector.After the recombined plasmid was transformed into component BL21, the His-tagged-STAT4 (565-748 amino acids) fusion protein was induced and obtained after denaturation,refolding,purification and dialysis.Conclusion:The eukaryotic expression vector containing the truncated human STAT4 gene encoding 565-748aa peptide has been successfully constructed and the fusion protein was obtained.