Expression and purification of milligram levels of inactive G-protein coupled receptors in E. coli

Abstract

G-protein coupled receptors (GPCRs) are seven transmembrane helical proteins involved in cell signaling and response. They are targets for many existing therapeutic agents, and numerous drug discovery efforts. Production of large quantities of these receptors for drug screening and structural biology remains challenging. To address this difficulty, we sought to express genes for several human GPCRs in E. coli. For most of the receptors, expression was poor, and was not markedly improved even in strains designed to compensate for differences in codon bias between human and E. coli genes. However, the gene for human NK 1 receptor (hNK 1 R) was expressed in large quantities as inclusion bodies in E. coli. The inclusion bodies were not soluble in chemical denaturants such as guanidine chloride or urea, but were soluble in ionic detergents such as SDS, and the zwitterionic detergent fos-choline. Using immobilized metal affinity chromatography, we purified milligram amounts of hNK 1 R. Although inactive in ligand binding assays, purified hNK 1 R in fos-choline micelles appeared to have a high content of alpha-helix, and was wellbehaved in solution. Thus this protein is suitable for additional biophysical characterization and refolding studies.

High-yield production, refolding and a molecular modelling of the catalytic module of (1,3)-β-d-glucan (curdlan) synthase from Agrobacterium sp.

“…For example, human antibody fragments [63], morphogenetic proteins [60], G-protein coupled receptors (GPCRs) [64] and glutamate receptors [65] have been refolded using highthroughput refolding trials and fractional folding screens [66]. Often, efficient on-column refolding techniques have allowed simultaneous renaturation and purification of proteins, where misfolded proteins refolded after their aggregation was prevented by immobilization [67,68].…”

An efficient production and characterization of HIV-1 gp41 ectodomain with fusion peptide in Escherichia coli system

“…Yield of overexpressed membrane proteins in E. coli system is typically low (Wagner et al, 2007), usually in the range of 5-15 mg/L culture (Bane et al, 2007;Imai et al, 2007;Lovering et al, 2007;Li et al, 2003). The yield of previous preparations of gp41 ectodomain recombinant protein lacking FP ranged from 20 to 60 mg/L culture.…”

New Techniques to Express and Crystallize G Protein‐Coupled Receptors

“…Further modifi cation to improve upon the MBP fusion system has involved the generation of a triple -protein fusion construct (MBP -GPCRthioredoxin [Trx]) that appears to further stabilize the receptor, and improve expression and purifi cation [66 -68] . Other factors infl uencing the expression levels are the E. coli strains used and the growth temperature [62,67,69,70] .…”

“…Moreover, the generation of inclusion bodies is poorly understood; despite the assumption that the expression of large hydrophobic proteins should readily form inclusion bodies, this is not always the case. A number of techniques have been developed to facilitate GPCR expression in inclusion bodies, including the use of strong promoters and high -copy -number plasmids [69,70] (pGEX and pET types), codon optimization [61] , fusions to ketosteriod isomerase [75] (which is commonly used to direct the formation of inclusion bodies), and glutathione -S -transferase [76] (known to drastically affect protein expression levels), yet there is no general strategy for the systematic production of GPCRs from inclusion bodies. Expression studies have been able to improve the levels of expression of GPCRs in inclusion bodies; however, this is only the fi rst step in the purifi cation pathway, and further steps involving protein solubilization, purifi cation, and renaturation are required.…”