Neurotensin Receptor 1: Purification by PoLiPa

Generating purified, stable and functional GPCR preparations

We used the neurotensin receptor 1 (NTSR1) to demonstrate the effectiveness of our PoLiPa GPCR purification process.

Challenge

The purification of solubilised G protein-coupled receptors (GPCRs) is notoriously challenging, which limits their access to structural/biophysical techniques that can accelerate drug discovery. Techniques such as thermostabilising mutations have been used successfully in some cases, but the process is time-consuming and has to be adapted to each GPCR.

Solution

Domainex has established a generic platform to generate any purified GPCR without the need for thermostabilising mutations or detergents. This was achieved using Polymer Lipid Particle (PoLiPa) technology to stabilise GPCRs by encapsulating them in a polymer that also encloses a small disc of native cell membrane lipids.

Neurotensin Figure 1 — **Figure 1:** Schematic representation of Domainex’s PoLiPa GPCR purification process. The target receptor is coloured orange (other membrane proteins are crimson). Cells expressing the receptor are solubilised with polymer (green) which creates PoLiPa with the proteins encapsulated within a ring of the original membrane lipids (grey). The target receptor is then isolated using protein purification techniques.

We used the neurotensin receptor 1 (NTSR1) to demonstrate the effectiveness of the approach.

Expression

Human NTS1R was expressed in mammalian HEK293 cells. The receptor was fluorescently labelled and a 6xhis tag included. Cell surface expression was confirmed by imaging.

Neurotensin Figure 2 — **Figure 2:** Fluorescence microscopy of clonal HEK293 cell line overexpressing NTSR1. Cell surface receptors are labelled by N-terminal tagging of the receptor with a fluorescent dye (red).

Solubilisation

Cells were solubilised with SMA2000 at room temperature. Soluble (PoLiPa containing NTS1R in native membrane) and insoluble fractions were separated by centrifugation.

Neurotensin Figure 3 — **Figure 3:** Left: structure of styrene maleic acid co-polymer (SMA). Right: A suspension of HEK293 cells overexpressing NTSR1 before and after solubilisation with SMA.

Purification

Purification of the PoLiPa-NTS1R was achieved by Nickel-NTA (NiNTA) affinity, followed by size exclusion chromatography. A purity of >75% and a yield of >200µg was achieved.

Neurotensin Figure 4 — **Figure 4:** Left: Native-PAGE gel with Coomassie staining over PoLiPa-NTSR1. Red arrow highlights the specific receptor band. Right: The same gel fluorescently imaged showing the receptor post-translationally labelled with fluorescent dye.

Coomassie staining showed partially purified protein (red arrow). Fluorescence detection showed a pure homogenous population of labelled receptor.

Validation

A novel ligand-observed, label-free LC-MS assay was used to pharmacologically validate the purified GPCR. A small molecule antagonist (SR-142948; Figure 5) was used as a tracer ligand. SR-142948 binding (Kd = 8.8nM) indicated that PoLiPa-NTS1R was folded and pharmacologically intact. Specific binding sites could be quantified using competition with an excess of another receptor antagonist.

Neurotensin Figure 5 — **Figure 5:** The structure of SR-142948

Neurotensin Figure 6 — **Figure 6:** Ligand-observed LCMS was used to pharmacologically validate correctly folded receptor binding sites. The small molecule NTSR1 antagonist SR-142948 was quantified using LC-MS. Saturation binding analysis using this approach to measure bound ligand shows high affinity binding (in line with literature reports) and low non-specific binding (by displacement with an additional antagonist SR-48692).

Neurotensin Figure 7 — **Figure 7:** Saturation binding analysis (LC-MS) of the NTSR1 antagonist SR-142948 to isolated membranes expressing NTSR1 or PoLiPa-purified NTSR1. Kd values are comparable between the two preparations suggesting that a similar pharmacologically intact protein is presented in each system.

Saturation binding analysis against PoLiPa-NTSR1 was used to determine the stability during storage (Figure 8). The purified receptor was found to be very stable even with prolonged storage.

Neurotensin Figure 8 — **Figure 8:** PoLiPa-NTSR1 is stable with prolonged storage. Saturation binding analysis by LCMS was used to quantify the concentration of receptor binding sites (left) and antagonist affinity (right) over time after storage at 4 or -80 °C. Receptor binding sites decrease gradually over 80 days. However, accurate affinity determination remains unchanged throughout.

Conclusion

The PoLiPa technology developed by Domainex allows the preparation of purified soluble GPCR complexes that can be used in biochemical, biophysical and structural studies. Ligand-observed LCMS is a very useful label-free assay technique for characterising ligand-binding to PoLiPa-GPCR reagents.

The PoLiPa-GPCR reagents are very stable and can be used over a prolonged period of several months, in contrast to other preparations. This approach is fast and generic, and in these regards, is superior to methods such as detergent-solubilisation and thermostabilising mutagenesis that have been developed to achieve similar ends.