【摘要】 大肠杆菌高密度发酵以包涵体形式表达融合蛋白Trx-rPA,表达量22%。包涵体蛋白洗涤后经金属螯合层析纯化,纯度达80%以上。经胱氨酸衍生,以脉冲加样形式复性,复性率可高达30%。经ETI-Sepharose纯化,复性的融合蛋白生物活性可达3.5×105IU/mgPr.。融合蛋白可被rEK酶切释放rPA,酶切效率达85%以上。酶切液经IDA-Sepharose和SP-Sepharose层析纯化,rPA纯度达98%以上,生物活性50万IU/mgPr.。1L发酵液经分离、复性及纯化后,可得高纯度rPA300mg以上。更多还原

【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 3.5×10 5 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.更多还原