Expression and purification of milligram levels of inactive G-protein coupled receptors in E. coli.
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 Escherichia 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 well-behaved in solution. Thus this protein is suitable for additional biophysical characterization and refolding studies.
Bane
Steven E
SE
Department of Chemical Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA.
Velasquez
Javier E
JE
Robinson
Anne Skaja
AS
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D008024:Ligands; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018040:Receptors, Neurokinin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins