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

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.