炭疽水肿因子在大肠杆菌中高效表达及一步法纯化

Efficient expression of anthrax edema factor in Escherichia coli and its single-step purification

摘要:

[目的] 本研究旨在建立一种简单快捷的炭疽水肿因子(EF)重组表达及纯化方法。[方法] 构建GST-EF融合表达载体,基于EF基因的密码子使用偏好,选择菌株Escherichia coli BL21-CodonPlus(DE3)-RIL为表达宿主,对EF进行诱导表达;细胞透性技术分离粗蛋白,进而利用亲和层析一步法纯化EF;Native-PAGE、竞争性抑制实验及cAMP浓度分析用于鉴定EF的生物活性。[结果] 实现了EF可溶性高效表达,透性化处理可有效抽提可溶性重组蛋白;利用亲和层析一步法纯化得到了纯度达96%的EF;EF可与保护性抗原(PA)结合形成水肿毒素,该毒素能够急剧提高CHO-K1细胞中cAMP的浓度。[结论] 本研究建立了一种高效快速制备具有生物活性的炭疽水肿因子的方法,为炭疽相关研究工作提供了新的选择。

关键词:炭疽;水肿因子;重组表达;亲和层析

Abstract:

[Objective] To develop a new method for efficient expression and rapid preparation of biologically active anthrax edema factor (EF). [Methods] EF was fused with GST and expressed in the host E. coli BL21-CodonPlus (DE3)-RIL by IPTG induction. The crud protein was extracted by permeabilization, and then EF was purified by onestep affinity chromatography. cAMP assay, Native-PAGE and competitive inhibition analysis were carried out to evaluate EF's biological activity. [Results] EF was expressed in soluble form and then purified to 96% purity by single-step. The recombinant EF was able to bind furin-nicked protective antigen (PA) to form edema toxin, which could elevate the intracellular cAMP level of CHO-K1 cells dramatically. [Conclusion] This work provides a timesaving method for purification of EF with high purity and good biological activity, which might be valuable for anthrax-related study.

Key words:anthrax;edema toxin;recombinant expression;affinity chromatography