Evaluation of Cell-free Expression System for the Production of Soluble and Functional Human GPCR N-formyl Peptide Receptors

Author(s): Xiaoqiang Wang, Jiqian Wang, Baosheng Ge.

Journal Name: Protein & Peptide Letters

Volume 20 , Issue 11 , 2013

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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.

Keywords: Biological function, cell-free expression system, detergent, G protein-coupled receptors, N-formyl peptide receptors, secondary structure.

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Article Details

VOLUME: 20
ISSUE: 11
Year: 2013
Page: [1272 - 1279]
Pages: 8
DOI: 10.2174/09298665113209990043
Price: $65

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