大肠杆菌表达Trx-rPA融合蛋白的复性纯化

Refolding the Fusion Protein of Trx-rPA

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3.5×10⁵ IU/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rPA 300mg以上。

关键词:瑞特普酶，复性，包涵体，融合蛋白

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.

Key words:Reteplase，rPA，Trx-rPA，Refolding