Refolding the Fusion Protein of Trx-rPA

Abstract:

Thioredoxin-rPA (Trx-rPA) was expressed in E.coli as inclusion body by high density fermentation.After washed，the inclusion body was dissolved in 6 mol/L Guanidine and 100 mmol/L DTT，which was adjusted to pH3.0 and dialysed against cold distilled water.After the fusion protein was purified by metal chelating affinity chromatography，its purity was up to 80%.By cystine derivatizing and pulse refolding，the yield of refolded Trx-rPA，of which the specific activity was around 3.5×10⁵ IU/mgPr.，was above 30%.Up to 85% of the refolded fusion protein could be cleaved by rEK. rPA，released from the breakage of fusion protein，was purified to homogeneity (purity ≥98%) by two-step purification of IDA-Sepharose and SP-Sepharose chromatography.Its specific activity was 580000IU/mgPr.The yield of rPA was above 300mg/L broth.