Chaperonin-enhanced Escherichia coli cell-free expression of functional CXCR4

• Synergies of the bacterial chaperonin GroEL-GroES and cell-free expression for the production of functionally folded CXCR4 is studied.
• GroEL-GroES greatly increases the rate and yield of CXCR4 functional folding.
• The structural stability and ligand binding affinity of CXCR4 can be improved with supplied GroEL-GroES.
• The cooperation between GroEL and GroES is required to promote efficient folding.
• New insights into membrane protein production and folding, as well as the role of molecular chaperones.

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 ∼1.1 mg/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.