Production of a G-protein Coupled Receptor (GCPR) Protein to Assess Target engagement

The client wanted to assess binding of test molecules to a membrane-spanning GPCR.  A suitable construct of this target protein was not commercially available with limited literature precedence. Production of membrane proteins for biophysical assays is a challenge due to low yields, low stability and the requirement for stable and functional extraction from the cell membrane.

Recruited our membrane protein expertise – Our team of membrane protein experts reviewed the target from a structural perspective, designed an optimal construct along with associated expression and purification methodologies.

Holistic protein design – Informed construct design to ensure the protein was active and fit for all downstream purposes.

Tailored purification strategy – Use of detergent and additives using two-step purification to ensure high purity.

Bespoke activity assay – design of a dye-based thermal shift assay suitable for challenging membrane proteins.

Using our team’s extensive experience in membrane protein production, we designed a construct with a soluble fusion domain, a C-terminal truncation and two affinity tags (FLAG and deca-Histidine) tag to aid purification. The protein was then expressed in insect cells using baculovirus transduction.

We developed a bespoke purification strategy with solubilization using LMNG detergent in the presence of the cholesterol analogue (CHS), followed by downstream purification using both anti-FLAG and NiNTA.

Using Differential Scanning Fluorimetry (DSF) we showed that the purified protein was functional. Typical DSF assays use a dye that becomes fluorescent when interacting with hydrophobic amino acids that are exposed upon the unfolding of a soluble protein. Since this was a hydrophobic membrane protein, an alternative strategy was adopted. Using our expertise coupled with the literature, we selected a dye which shows an increase in fluorescence signal upon reaction with with cysteines that are only exposed once the membrane protein unfolds. These data showed an increase in thermal stability (via an increase in melting temperature) in the presence of a know ligand, indicating that the protein was functional.

• Detergent solubilization and purification using Ni2+ affinity and Size Exclusion Chromatography resulted in pure, monodispersed protein (Figure 1).

• This purified GPCR was active, as assessed by thermal shift analysis using a known lipidic ligand (Figure 2).

Ligand binding showed that the protein was pure, functional and suitable for downstream assays.

We delivered high-quality, functional protein:

• High purity, monodispersed GPCR protein production from insect cells.
• Protein assessed as functional.
• Enabled the assessment of tool compounds using biophysical methods.