title: Evaluation of cell-free expression system for the production of soluble and functional human GPCR N-formyl peptide receptors.
abstract: Human N-formyl peptide receptors (FPRs) belong to the G protein-coupled receptors (GPCRs) superfamily, the most frequently addressed drug targets in the pharmaceutical industry, and are considered to play important roles in innate immunity and host defense mechanisms. Although still a highly challenging task, the availability of soluble and functional GPCRs including FPRs in milligram quantities is essential to spur the advancement of protein-based structural and functional studies for drug discovery. In this report, the applicability of E. coli extracts-based cell-free expression system to producing soluble and active human FPRs and hence to FPRs protein-based research was evaluated, during which human FPR3 was selected as our prototype receptor. To better solubilize the freshly expressed human FPR3, a panel of different detergents (mostly nonionic detergents) were selected and evaluated in the cell-free system devoid of natural membrane. After one-step immunoaffinity purification, the secondary structure and biological function of purified FPR3 were characterized. A final yield of 0.6 mg functional human FPR3 per ml reaction volume was obtained. The demonstrated proper folding and functionality of the cell-free produced human FPR3 opens a new avenue for the fast and efficient generation of human FPRs (and even other GPCRs) for structural and functional analysis.
authors.affiliation: State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao Economic Development Zone, Qingdao 266580, P.R. China. wangxq001@upc.edu.cn.
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Wang
authors.forename: Jiqian
authors.initials: J
authors.lastname: Wang
authors.forename: Baosheng
authors.initials: B
authors.lastname: Ge
mesh_terms: D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D017433:Protein Structure, Secondary; D044042:Receptors, Formyl Peptide / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: C469985:FPR3 protein, human; D044042:Receptors, Formyl Peptide; D043562:Receptors, G-Protein-Coupled