Expression, Purification And Characterization Of Non-taged Recombinant Human Thioredoxin

BackgroundThioredoxin (Trx) is a heat stable, acidic small protein (Mr12,000) with a conserved active site sequence Trp-Cys-Gly-Pro-Cys that catalyzes many redox reactions through the reversible oxidation of its active site dithiol to a disulfide. Trx fulfills a variety of biological functions, such as the control of gene expression, cell proliferation and cell death, the elimination of reactive oxygen species in the cell, the inhibition of neutrophil activation and neutrophil migration. We all known that Trx most intensively in human disease. Trx alterations have been implicated in cataract formation, cancers, AIDS, rheumatoid arthritis, complications of diabetes, hepatic and renal diseases etc. Also there has evidence show that Trx functions as a critical myocardial protective factor directly via antioxidant effects and indirectly by protein-protein interaction with key signaling molecules.Now experiment research used Trx, which has complex process of preparation product and expensive price. So it is greatly limited for extensive clinical trials and usage. To establish a simple and inexpensive process of Trx preparation is critic for widely used in clinic.Therefore, how to explore a feasible and economic effective technology to purification, production rhTrx and realize a great quantity of preparation rhTrx that was we try to solve.Aims1. To construct prokaryotic expression system for mass production of recombinant human thioredoxin; To detect the stability of biological characteristics of recombinant E. coli BL21/pET-22b(+)-rhTrx expressing human thioredoxin; To establish the expression, purification process and Preliminary quality procedures of thioredoxin.2. Utilize The insulin disulfide reduction assay analysis the activity of rhTrx In vitro and investigate the effect of rhTrx in mice after myocardial ischemia/reperfuion (MI/R) injury.Methods1. 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.2. Recombinant E. coli BL21/pET-22b(+)-rhTrx was subcultured for50passages, and subjected to transmission electron microscopy, Gram staining and biochemical examination as well as test for expression level of target protein and property of plasmid every10passages.3. 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 for identification and purity assay respectively.4. The insulin disulfide reduction assay was set to analysis the activity of rhTrx In vitro. Set a model of myocardial ischemia/reperfusion (I/R) injury and administered with rhTrx before reperfusion to analysis the activity of rhTrxin vivo.Results1. Gene sequencing demonstrated that thioredoxin coding sequence was cloned into pET-22b(+) vector successfully.2. The results of restriction analysis and sequencing of various passages were in agreement. All the expression levels of target protein in recombinant E. coli BL21/pET-22b(+)-rhTrx of passages0,10,20,30,40and50passages were about20%, which showed no significant difference. All the passages were negative in Gram staining and showed typical characteristics of E. coli under transmission electron microscope and in biochemical examination.3. The prokaryotic expression system we constructed achieved high yield of thioredoxin (～180mg/5L of fermentation broth), which was identified by Western blotting and MALDI-TOF-MS, with an estimated the molecular weight of12,000. The purity of thioredoxin is more than95%.4. In vitro, the rhTrx could reduce the disulphide bonds of insulin. And in vivo, rhTrx could significantly reduce I/R-induced myocardial injury and apoptosis.Conclusions1. The prokaryotic expression system we constructed could achieves mass production of recombinant human thioredoxin; The recombinant E.coli BL21/pET-22b(+)-rhTrx shows stable biological characteristics and might be used as a bacterial seed for production; can be highly purified by two-steps ion exchange chromatography. Our preliminary study provides the foundation for the large-scale industrial production of thioredoxin.2. The purified rhTrx have high activity, And will be used as a new therapeutic for antagonist myocardial ischemia-reperfusion injury.