培养基pH值与提纯方法对大肠杆菌表达rhGM-CSF产率的影响

The effects of medium pH and purification method on product rate of expression rhGM-CSF in recombinant E.coli

目的为了提高大肠杆菌表达的重组人粒细胞 巨噬细胞集落刺激因子 (rhGM CSF)的产率 ,对影响工程菌生长、产物表达和产物提纯等因素进行探索。方法设计 3种pH值培养基 ,以菌得率、表达率为指标 ,观察 pH对工程菌生长和表达的影响。以包涵体得率、纯度和产品得率、纯度、活性为指标 ,观察表达产物提纯各步对产率的影响。用SDS PAGE薄层扫描法测定包涵体和产品纯度。用TF1细胞MTT法测定产品rhGM CSF活性。结果 pH7.0培养基最好 ,菌得率 2 .2 2g/L ,表达率为 2 3 % ,比其它pH值培养基约高 1倍。菌体不经冻存处理即进行破菌制备包涵体 ,得率可提高 2 .5倍。在rhGM CSF复性步中 ,复性液中加入少量PEG 40 0 0 ,可提高复性率 0 .8倍。采用优化条件制备大肠杆菌rhGM CSF ,可提高产率约 8倍。结论影响大肠杆菌表达rhGM CSF产率的因素较多 ,优化培养、表达、纯化各步的条件 ,均可大幅度提高产率。

PurposeThe aim is to study the influence factors of recombinant E.coli cell growth,product expression and purification,in order to raise the product rate of rhGM CSF.MethodsThree pH media were planed to observe the effects of pH on E.coli cell growth and expression with the indexes of the cell density and expression level.The influences of various treatments on product rate were observed with the indexes of the volume and purity of inclusion bodies and the yield,purity,activity of product.Using SDS PAGE method for analysis of purity.The product bioactivity was determined by using TF1 cell and MTT colorimetric method.ResultsThe cell density in the medium pH 7.0 was 2.22 g/L;the product expression rate was 23%.It was two times higher than other pH media.The inclusion body prepared by bacteria without freezing store was 2.5 times volume than freezing store.During renatura tion PEG 4000 could raise 0.8 times active product.The optimum conditions used rhGM CSF product rate could raise 8 times.ConclustonThe product rate of rhGM CSF expressed in E.coli was affected by many factors.The optimum condition for culture,expression and purification could raise quite a number of product rate.