环状排列黄色荧光蛋白在大肠杆菌及毕赤酵母中表达的比较研究

Comparison of circularly permuted yellow fluorescent protein from Escherichia coli and Pichia pastoris

采用基因工程方法,实现了cpYFP在毕赤酵母X33的表达,并与大肠杆菌BL21(DE3)表达做对比.结果表明,毕赤酵母胞外分泌的目的蛋白不仅背景较大肠杆菌表达的清晰,且荧光强度为前者的3倍.同时,毕赤酵母表达的重组cpYFP对超氧阴离子自由基反应的灵敏性高于大肠杆菌表达的cpYFP.这些结果表明,毕赤酵母表达体系可作为制备重组cpYFP的良好体系,也为cpYFP在自由基生物学领域的广泛应用提供充足的原料来源.

The circularly permuted yellow fluorescent protein （cpYFP）,which acts as a superoxide sensor,is mainly expressed and obtained by Escherichia coli.However,the prokaryotic system has many deficiencies,such as producing endotoxin and failing to modify target protein.In order to cover these disadvantages,Pichia pastoris （X33）,a typical eukaryotic microorganism,was used to express cpYFP for the first time in this study.Comparing with E.coli.（BL21 （DE3）） expression system,it was found that the amount of cpYFP secreted into the medium of P.pastoris （X33） was 3 folds of that in E.coli and the by-products was fewer.Furthermore,cpYFP derived from Pichia showed a higher sensibility to superoxide.These results suggests that Pichia is an efficient system for the expression of recombinant-cpYFP,providing sufficient material for the use of cpYFP in the field of free radical biology.