基因重组Echistatin发酵工艺的优化

目的:利用基因工程方法对一种蛇毒锯鳞蝰素蛋白的发酵纯化工艺进行优化,以提高目的蛋白的产量和纯度。方法:对工程菌进行发酵培养并诱导表达,研究不同的培养基、不同补料方式、溶解氧浓度、培养和诱导时间对工程菌产量和目的蛋白表达量的影响,利用几丁质亲和层析纯化Ecs融合蛋白,通过合适温度和pH裂解融合蛋白得到Ecs纯品,并鉴定和检测Ecs活性。结果:经过高密度发酵优化后,菌体湿重可达110g/L,目的蛋白表达量约占总蛋白的40%;亲和层析纯化后,得到Ecs单体,得率为68mg/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。结论:通过发酵和纯化工艺优化,大大提高了目的蛋白产量,为进一步规模化研究和生产奠定了基础。

Optimization of High-density Fermentation of Recombinant Echistatin

Objective:To improve the productivity and purity of Echistatin protein expressed in E.coli by optimizing the fermentation process. Methods:The recombinant bacteria were cultured by fermentation and IPTG induction. The fermentation parameters were analyzed by observing the influence on recombinant protein expression and cell growth with the change of medium, feeding method, dissolved oxygen, culture and induction time. The fusion protein was purified by chitin affinity chromatography. The bioactivity of Ecs was determined after fusion protein was cleaved by DTT under proper temperature and pH. Results:After optimization of high-density fermentation, the derivative of recombinant fusion protein was about 40% of total protein and 110 g of wet bacteria per liter was obtained. Ecs was purified by affinity chromatography. The recovery rate of the final product was about 68 mg/L fermentation volume. Biological activity assay showed that purified Ecs could inhibit the aggregation of platelet in vivo with similar bioactivity to wild Ecs. Conclusion:The fermentation conditions of Ecs fusion protein was optimized. It could improve the productivity of Ecs protein, and lays the foundation for the large-scale production of Ecs.