趋化因子受体CXCR4b在大肠杆菌中的高效表达、增溶及纯化

High-level Expression, Solubilization and Purification of Chemokine Receptor CXCR4b in Escherichia coli

摘要:

膜蛋白在细胞分化、信号转导等生理活动中发挥着重要作用, 然而膜蛋白结构与功能的研究却受到高质量蛋白制备的严重制约。将斑马鱼趋化因子受体CXCR4b基因克隆到pMAL-p4x表达载体中, 在大肠杆菌TB1中表达麦芽糖结合蛋白(MBP)-CXCR4b融合蛋白。通过系统优化其发酵表达条件, 实现了CXCR4b的过量表达。最佳表达条件为: 宿主菌选用大肠杆菌TB1, TB培养基, 诱导剂IPTG浓度为0.5 mmol/L, 诱导时机为对数中后期。通过对10种不同表面活性剂的筛选, 发现DM、FC-14和Brij35等表面活性剂对CXCR4b有较好的增溶效果。利用Ni2+亲和色谱和S200凝胶色谱两步纯化, 得到CXCR4b的纯度可达90%以上。圆二色谱检测显示纯化的CXCR4b呈典型的α螺旋结构。

Abstract:

Membrane protein plays a vital role in cellular differentiation and signal transduction. However, the study of structure and function of membrane protein is still limited due to lack of the efficient preparation for enough high quality membrane proteins. In this paper, zebrafish CXCR4b gene was cloned into pMAL-p4x vector, then was expressed as a fusion of MBP-CXCR4b in E. coli TB1. Over-expression of CXCR4b was achieved after optimizing expression conditions systematically. The optimal expression conditions were obtained as: E. coli TB1 as host strain, TB medium, concentration of IPTG 0.5 mmol/L, inducing at the late mid-logarithmic growth phase. Totally 10 surfactants were screened for their ability to successfully solubilize CXCR4b from cell membranes. As a result, DM, FC-14 and Brij35 exhibited good solubizing ability for CXCR4b. CXCR4b was then purified using Ni2+ chelating affinity chromatography followed with gel filtration S200. The purified protein showed correct apparent molecular weight on SDS-PAGE and the purity was up to 90%. A typical α helical structure was determined with circular dichroism analysis, which means that the purified CXCR4b had folded into a reasonable conformation.