Chaperonin-enhanced Escherichia coli cell-free expression of functional CXCR4.
G protein-coupled receptors (GPCRs) are important therapeutic targets for a broad spectrum of diseases and disorders. Obtaining milligram quantities of functional receptors through the development of robust production methods are highly demanded to probe GPCR structure and functions. In this study, we analyzed synergies of the bacterial chaperonin GroEL-GroES and cell-free expression for the production of functionally folded C-X-C chemokine GPCR type 4 (CXCR4). The yield of soluble CXCR4 in the presence of detergent Brij-35 reached ∼1.1mg/ml. The chaperonin complex added was found to significantly enhance the productive folding of newly synthesized CXCR4, by increasing both the rate (∼30-fold) and the yield (∼1.3-fold) of folding over its spontaneous behavior. Meanwhile, the structural stability of CXCR4 was also improved with supplied GroEL-GroES, as was the soluble expression of biologically active CXCR4 with a ∼1.4-fold increase. The improved stability together with the higher ligand binding affinity suggests more efficient folding. The essential chaperonin GroEL was shown to be partially effective on its own, but for maximum efficiency both GroEL and its co-chaperonin GroES were necessary. The method reported here should prove generally useful for cell-free production of large amounts of natively folded GPCRs, and even other classes of membrane proteins.
State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, Qingdao 266580, PR China. Electronic address: haixiafengye@126.com.
Haixia
H
Chi
State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, Qingdao 266580, PR China; College of Science, China University of Petroleum (East China), Qingdao 266580, PR China. Electronic address: wangxq001@upc.edu.cn.
Xiaoqiang
X
Wang
State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, Qingdao 266580, PR China. Electronic address: ljq325@163.com.
Jiqiang
J
Li
State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, Qingdao 266580, PR China. Electronic address: renh@upc.edu.cn.
Hao
H
Ren
State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, Qingdao 266580, PR China. Electronic address: fhuang@upc.edu.cn.
Fang
F
Huang
D018835:Chaperonin 10 / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D019718:Receptors, CXCR4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C527276:CXCR4 protein, human; D018835:Chaperonin 10; D018834:Chaperonin 60; D019718:Receptors, CXCR4; D011994:Recombinant Proteins
Biological activity; Cell-free expression; Chaperonin; G protein-coupled receptor; Protein folding; Structural stability