High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1 Hui Ren 1 , Daoyong Yu1,2, Baosheng Ge1,2, Brian Cook 1 , Zhinan Xu 1 , Shuguang Zhang 1 * 1 Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 2 Center for Bioengineering and Biotechnology, China University of Petroleum, Qingdao, Shandong, People’s Republic of China Abstract Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis. Citation: Ren H, Yu D, Ge B, Cook B, Xu Z, et al. (2009) High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1. PLoS ONE 4(2): e4509. doi:10.1371/journal.pone.0004509 Editor: Yann Astier, Instituto de Tecnologia Qu´ımica e Biolo´ gica, Portugal Received October 4, 2008; Accepted November 21, 2008; Published February 18, 2009 Copyright: 2009 Ren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: SZ gratefully acknowledges a John Simon Guggenheim Fellowship to provide freedom to pursue frontiers of studying membrane protein structures. This work is supported in part by NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: shuguang@mit.edu Introduction G-protein-coupled receptors (GPCRs) primarily function as cell- surface receptors responsible for the transduction of extra-cellular stimuli into intra-cellular signals by binding extra-cellular ligands including photons, ions, lipids, peptides, nucleosides, nucleotides, neurotransmitters and peptide hormones. Structurally, they share a common hydrophobic core composed of seven-transmembrane a-helices (7TM) [1,2]. Approximately 4% of human genes code for GPCRs and by the current count there are ,800 functional genes. They comprise the largest superfamily of human integral membrane proteins [3,4]. GPCRs play vital roles in a wide range of biological processes and are involved in a remarkable array of signaling events ranging from memory, sight, and smell to sexual development and the regulation of blood pressure [5,6]. Therefore, GPCRs are attractive therapeutic targets for drug design. Currently, about 50% of pharmaceutical drugs target GPCRs [3]. Despite their critical importance, our current understanding of structure and function of GPCRs is inadequate because of their low natural abundance. Thus, for structural studies, which require milligram quantities of purified membrane protein [7], production in heterologous systems is required, but has been extremely difficult to accomplish. Up to now the molecular structures of only 5 unique GPCRs have been determined including bovine rhodopsin with and without the retinal ligand as well as with a C-terminal 11-residue peptide fragment of a Ga-protein (Ga-CT) [8,9,10]; a highly engineered human b2-adrenergic receptor with a replaced intracellular loop 3 (IC3) [11,12], and a turkey b1-adrenergic receptor with the IC3 domain partly removed and most C-terminus deleted [13]. Currently not a single chemokine receptor structure is known. Determination of the molecular structures of GPCRs including chemokine receptor still remains an enormous challenge, largely due to the notorious difficulty to obtain large quantities of purified proteins. The same is true for other membrane proteins. This is evident also from the fact that there are only 178 unique membrane protein structures among 410 membrane protein structures from over 54,000 structures available in the current Protein Data Bank http://www.rcsb.org/pdb/home/home.do (November 2008). PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4509