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

Abstract

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 fromE. colicells 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 cell-free 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 fromExiguobacterium sibiricum(ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein fromB. subtilisallows 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.

Polar Interactions Trump Hydrophobicity in Stabilizing the Self-Inserting Membrane Protein Mistic

“…In 11 reports, 13,15,[17][18][19][20][21]23,25,27,28 a total of 23 detergents were screened for their solubilization efficiencies (Supporting Information Table S4). Systematic solubilization trials 25 using a homologous series of phosphocholine detergents showed that protein yield increases with chain length, mirroring the dependence of conformational stability on micelle thickness (Figure 4a).…”

“…For instance, the membrane pore of staphylococcal α-hemolysin remains functional after truncation of large parts of its transmembrane domain, and amphipathic peptides rich in cationic and anionic residues may form membrane-spanning charge zippers . An even more drastic example is Mistic, an unusual Bacillus subtilis protein that is essential for biofilm formation and is biotechnologically exploited as a fusion tag to support the membrane targeting, insertion, and detergent-mediated solubilization of other membrane proteins. ,− Mistic comprises 110 residues that are arranged into a four-helix bundle exposing numerous polar and charged residues (Supporting Information Figure S1), which is incompatible with a transmembrane topology. Having a predicted pI of 4.5, a net charge at pH 7.0 of −12, and a mean hydrophobicity on the normalized Kyte/Doolittle scale of 0.43, Mistic should be more hydrophilic than typical soluble globular proteins and might even be expected to be an intrinsically disordered protein .…”

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

“…As mentioned above, fusion tags can have different functions including enhancing heterologous protein expression when placed at the N-terminus ( Haberstock et al, 2012 ; Ki and Pack, 2020 ). This allowed, for instance, to increase yields of a GPCR 5–38 times, resulting in sufficient protein amounts for structural-functional studies ( Lyukmanova et al, 2012b ). Most fusion proteins have added sequences encoding an affinity tag that can be added at either end of the cDNA; small tags may also be added by primer extension PCR.…”

Large-scale production and protein engineering of G protein-coupled receptors for structural studies

“…Expression efficiency in a cell-free system sometimes depends on an N-terminal tag used, with TrxA and some smaller tags, like T7-tag, being most successful ( Ishihara et al, 2005 ; Haberstock et al, 2012 ; Lyukmanova et al, 2012 ). This is explained by different secondary structure of the corresponding mRNA affecting the translation efficiency.…”