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 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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Bane SE,Velasquez JE,Robinson ASdoi
10.1016/j.pep.2006.10.017subject
Has Abstractpub_date
2007-04-01 00:00:00pages
348-55issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(06)00321-4journal_volume
52pub_type
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