瑞替普酶融合蛋白在大肠杆菌中表达条件的优化及其复性

Optimization of Expression Conditions for Reteplase Fusion Protein in Escherichia coli and its Renaturation

目的:为优化瑞替普酶(reteplase,r-PA)在大肠杆菌中的高效融合表达的条件,并提高其复性率。方法:通过改变诱导时间和温度、诱导剂浓度、培养基pH值及氨苄青霉素(Amp)浓度等条件,利用SDS-PAGE分析以上条件的改变对表达产物产量的影响。运用金属螯合层析对融合蛋白进行纯化和复性。结果与结论:LB培养基pH值为6,Amp浓度为100μg/mL,乳糖浓度为5 mmol/L的条件下39℃诱导4 h可获得高效表达的r-PA融合蛋白,其表达量占全菌蛋白的70%,其产物主要以包涵体形式存在。融合蛋白运用金属螯合层析一步纯化复性,其复性率可达10%,比活为55.7 U/μL。

AIM:To optimize the conditions for highly expressing reteplase(r-PA) in Escherichia coli,and to increase its renaturation efficiency.METHODS:By changing the induction time and temperature,the medium pH and the concentration of ampicillin,the effects of the experiment settings on the expression product were analyzed by SDS-PAGE and using IMAC to purify and renature the fusion protein.RESUTLS AND CONCLUSION:The results showed that the highly expressed r-PA fusion protein,which mainly existed in the inclusion body,was obtained in the optimal condition of 39 ℃,pH 6.0 of LB medium,100 μg/mL of ampicillin,5 mmol/L of lactose and induction for (4 h).The expression was 70% of the total protein.Additionally,the expressed protein was purified and renatured by IMAC,and the renaturation efficiency was 10%.Specific activity of the fusion protein was 55.7 U/μL by fibrin agarose plate assay.