Recombinant Expression, in Vitro Refolding, and Biophysical Characterization of the Human Glucagon-like Peptide-1 Receptor

Abstract

Activation of the glucagon-like peptide-1 receptor (GLP-1R) upon ligand binding leads to the release of insulin from pancreatic cells. This strictly glucose-dependent process renders the receptor and its ligands useful in the treatment of type II diabetes mellitus. To enable a biophysical characterization in vitro, we expressed the human full-length GLP-1R in the cytosol of Escherichia coli as inclusion bodies. After purification, refolding of the SDS-solubilized receptor was achieved by the exchange of SDS against the detergent Brij78 using an artificial chaperone system. Far-UV circular dichroism spectroscopic studies revealed that the receptor adopts a characteristic alpha-helical structure in Brij78 micelles. Ligand binding of the renatured protein was quantified by fluorescence quenching and surface plasmon resonance spectroscopy. In the presence of Brij micelles, the refolded receptor binds the agonist exendin-4 with an apparent dissociation constant of approximately 100 nM in a reversible one-step mechanism. To demonstrate that the detected ligand binding activity is not only due to an autonomously functional N-terminal domain (nGLP-1R) but also due to additional contacts with the juxtamembrane part, we separately expressed and refolded the extracellular domain relying on identical protocols established for the full-length GLP-1R. In support of the suggested multidomain binding mode, the nGLP-1R binds exendin-4 with a lower affinity (K(app) in the micromolar range) and a different kinetic mechanism. The lower ligand affinity of the nGLP-1R results entirely from a decreased kinetic stability of the receptor-ligand complex, dissociation of which is approximately 40-fold faster in the case of the nGLP-1R compared to the full-length GLP-1R. In summary, a framework was developed to produce functional human full-length GLP-1R by recombinant expression in E. coli as a prerequisite for eventual structure determination and a rigorous biophysical characterization including protein variants.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

“…reported the E . coli recombinant expression and in vitro refolding of functional hGLP1R, where K D value was unmeasurable for GLP-1 and determined to be ~180 nM for Ex-4 using fluorescence quenching and surface plasmon resonance methods [56], which is comparable to our results. On the other hand, a lower affinity can also be caused by (1) fluorescent labeling of the peptide ligands, (2) the non-native lipid environment and (3) absence of native receptor-affiliated proteins.…”

Polyalanine-independent Conformational Conversion of Nuclear Poly(A)-binding Protein 1 (PABPN1)

“…Coding fragments for truncated PABPN1 variants were amplified from this vector and ligated into pET SUMOadapt (29). Recombinant protein production was performed by fermentation in complex medium in a 10-liter bioreactor (BIOSTAT C-DCU; Sartorius Stedim) as described previously (30) with the following modifications: at A 600 ϭ 40 -50, protein expression was induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside. The cultivation temperature was reduced to 30°C to prevent inclusion body formation.…”

Calcium-Dependent Ligand Binding and G-protein Signaling of Family B GPCR Parathyroid Hormone 1 Receptor Purified in Nanodiscs

“…The receptor belongs to family B GPCRs. To date, there have been a small number of successful cases of purifying family B GPCRs, which include pituitary adenylate cyclase-activating peptide receptor purified from bovine brain 29 and insect cells 30 , PTH1R purified from COS-7 cells 33 , HEK293S cells 11 and E. coli 27 , glucagon-like peptide 1 receptor purified from both E. coli 42 and insect cells 49 , and two other receptors, corticotrophin-releasing factor receptors 1 and 2β, obtained using cell-free expression methods 18 . In this study, we choose PTH1R as our target receptor, because it has been shown to be stable in detergents.…”