N-Terminal Fusion Tags for Effective Production of G-Protein-Coupled Receptors in Bacterial Cell-Free Systems

G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts from E. coli cells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cellfree production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin from Exiguobacterium sibiricum (ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein from B. subtilis allows to increase the CF synthesis of the target GPCRs by 5–38 times, resulting in yields of 0.6–3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.