Expression,Purification and Characterization of Non-taged Recombinant Human Thioredoxin

Objective:To construct prokaryotic expression system for mass production of recombinant human thioredoxin and establish the purification process of thioredoxin.Methods: Total RNA was extracted from HEK293(human embryonic kidney cells).The thioredoxin coding sequence was subcloned into the pET-22b(+) vector after amplified by PCR.The recombinant plasmids were transformed into E.coli BL21(DE3),and the thioredoxin was expressed with IPTG induction.The expressed thioredoxin was purified by two-step ion exchange chromatography and tested by SDS-PAGE,Western blotting,MALDI-TOF-M,HPLC,and insulin disulfide reduction assay for identification,purity assay and activity determination,respectively.Results: Gene sequencing demonstrated that thioredoxin coding sequence was cloned into pET-22b(+) vector successfully.The prokaryotic expression system achieved high yield of thioredoxin(180 mg/5L of fermentation broth),which was identified by Western blotting and MALDI-TOF-MS,with an estimated the molecular weight of 12 000.The purity of thioredoxin is more than 95%.The activity of purified thioredoxin had the same activity as the standard control.Conclusion: The prokaryotic expression system could achieve mass production of recombinant human thioredoxin,which can be highly purified by two-steps ion exchange chromatography.This preliminary study provides the foundation for the large-scale industrial production of thioredoxin.