Prokaryotic expression, in vitro folding, and molecular pharmacological characterization of the neuropeptide Y receptor type 2

Abstract

G protein-coupled receptors (GPCRs) are a class of membrane proteins that represent a major target for pharmacological developments. However, there is still little knowledge about GPCR structure and dynamics since high-level expression and characterization of active GPCRs in vitro is extremely complicated. Here, we describe the recombinant expression and functional folding of the human Y(2) receptor from inclusion bodies of E. coli cultures. Milligram protein quantities were produced using high density fermentation and isolated in a single step purification with a yield of over 20 mg/L culture. Extensive studies were carried out on in vitro refolding and stabilization of the isolated receptor in detergent solution. The specific binding of the ligand, the 36 residue neuropeptide Y (NPY), to the recombinant Y(2) receptors in micellar form was shown by several radioligand affinity assays. In competition experiments, an IC(50) value in low nanomolar range could be determined. Further, a K(D) value of 1.9 nM was determined from a saturation assay, where NPY was titrated to the recombinant Y(2) receptors.

Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration

“…The expression yield was about 20 mg Y2 receptor per liter culture medium. For comparison, this Y2 receptor variant was also folded in vitro using the previous protocol for the wild type Y2 receptor (Schmidt et al , 2009 ). Minor increases in stability and folding yield could be observed for the cysteine deficient variant, but the long-term stability in high concentration remained poor (data not shown).…”

“…Next, the cDNA encoding the receptor variant was ligated into a bacterial expression vector and expressed recombinantly in E. coli in non-functional form and purified as described previously (Schmidt et al , 2009 ). The expression yield was about 20 mg Y2 receptor per liter culture medium.…”

“…These aggregates must be isolated from the cell paste, solubilized mostly using the ionic detergent sodium dodecyl sulfate (SDS) and transferred into a functional form by in vitro folding in micelle-or bicelle-like environments (Baneres et al , 2011 ). For several GPCRs, this strategy is feasible, resulting in reasonable amounts of functional receptor (Kiefer et al , 1996 ;Dahmane et al , 2009 ;Schmidt et al , 2009 ;Schimmer et al , 2010 ;Damian et al , 2012 ;Park et al , 2012a ). Nevertheless, structural studies often remain unsatisfactory due to the poor long-term stability of these preparations, forming aggregates or non-functional oligomers, especially at the required high concentrations.…”

“…That may cause two important problems for the in vitro preparation. (i) The free cysteine residues of GPCRs in micelles are not as wellprotected as in the cellular membrane and may, therefore, be prone to form intermolecular disulfide bonds leading to oligomeric structures (Schmidt et al , 2009 ). (ii) During in vitro folding, a redox-shuffling system based on reduced (GSH) and oxidized (GSSG) glutathione is usually used for the formation of the required disulfide bond (Baneres et al , 2011 ).…”

Towards improved receptor targeting: anterograde transport, internalization and postendocytic trafficking of neuropeptide Y receptors

“…In order to unravel the similarities and differences in Y receptor structures, and more precisely in their binding pockets, chimeric receptor approaches, peptide alanine-scans (Pedragosa Badia et al , 2013 ), recombinant receptor expression (Schmidt et al , 2009 ) and NMR studies were performed. Several peptide residues were identified as being involved in receptor binding, while determination of the receptor counterpart was more difficult.…”