蛇毒锯鳞蝰素融合蛋白的发酵与纯化

研究大肠杆菌表达重组蛇毒锯鳞蝰素(Echistatin,Ecs)融合蛋白的发酵和纯化工艺。将Ecs基因插入表达载体pTXB1,转化E.coliBL21(DE3)构建工程菌。对工程菌进行补料分批培养并诱导表达,研究培养基、培养和诱导时间对工程菌生长和目的蛋白表达的影响,几丁质亲和层析纯化Ecs融合蛋白,经DTT裂解后,检测Ecs活性。发酵后菌体湿重可达75g/L,融合蛋白表达量约占总蛋白的35%,重组质粒在BL21宿主菌中传代稳定。亲和层析纯化后,得到Ecs单体,得率为28mg/L发酵液。生物学活性分析显示,重组Ecs能有效抑制血小板的聚集,其活性与天然Ecs相似。优化了Ecs融合基因工程菌的发酵和纯化条件,为规模化生产奠定基础。

Fermentation and purification of Echistatin fusion protein expressed in Escherichia coli

Lots of studies of Echistatin (Ecs) have proved its wide use in many aspects. However, the low yield of Ecs has impeded the relative researches of the protein. To establish the high-level expression system of Ecs, the fermentation and purification process of Ecs fusion protein expressed in E. coli were optimized. The Ecs gene was introduced into vector pTXB1 and placed under the control of highly efficient T7 promoter system. The cloned Ecs gene was expressed in E. coli BL21 (DE3) as soluble form. The Ecs production and biomass accumulation were optimized by examining medium composition, point of induction and induction time in fed-batch fermentation. Biomass accumulation was greatly affected by medium gradient, reaching 50.3g/L in 2 x YT medium. Ecs production was found to increase to 35% of total protein with 75g/L biomass accumulation after induced for 4h. Purification of Ecs from supernatant of sonication was done using one-step chromatographic procedure with chitin affinity chromatography and DTF cleavage, resulting in yields of 28mg/L and > 90% purity. The bioactivity of purified Ecs was determined and the result showed that purified Ecs could inhibit the aggregation of platelet in vitro with similar bioactivity to wild Ecs. This optimized method is readily scaled up for the expression and purification of Ecs in sufficient quantities for further structural and biological studies and applications.