Simplifying G Protein-Coupled Receptor Isolation with a Calcium-Dependent Fragment Complementation Affinity System

Abstract

The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging because the process requires solubilization in detergent micelles and multistep affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents, and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly, and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB, in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics

“…A 37 amino acid EF2 affinity ligand coupled to agarose was then used in calcium-dependent fragment complementation-based pull down, using either radioimmunoprecipitation assay buffer (RIPA) buffer containing 150 mM NaCl and detergents deoxycholic acid (1%) and NP-40 (1%) or membrane solubilization buffer (MSB) that we have previously employed in the purification of EF1-tagged cannabinoid receptor CB 2 from Escherichia coli membranes and have adapted for the enrichment of EF1-tagged kinases from HEK293T cells. [3][4][5] MSB contains 30% glycerol, 200 mM NaCl and a cocktail of 0.5% 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate, 0.1% cholesteryl hemisuccinate tris salt and 1% n-dodecyl--D-maltoside that combine to disrupt cellular membranes to release integral membrane proteins and to stabi-lize these in detergent micelles. [6] Stabilized membrane proteins may as a result be retained in soluble cell lysate preparations for capture by affinity pull down rather than being removed by membrane sedimentation during centrifugation in preparative steps.…”

The nuclear transport receptor TNPO1 binds macrophage immunometabolism regulator MACIR via a PY-NLS motif

“…Identification of function of this protein in the cell then requires the careful identification of the protein:protein interaction networks it participates in and the fine mapping of interactions that regulate the localisation of the protein that may impact on the immunometabolism phenotypes. We have previously developed a calcium-dependent fragment complementation-based affinity pull down system that uses the highly specific recognition between two fragments, EF1 & EF2, of a small calcium binding protein to pull down overexpressed kinase proteins from HEK293T cells and 7 transmembrane proteins from Escherichia coli membranes using a membrane solubilizing buffer (MSB) that contains high concentrations of glycerol and a cocktail of detergents that disrupt cellular and intracellular membranes and stabilises membrane proteins in detergent micelles (3,4). In this study we employ this approach to identify MACIR interacting proteins from all compartments in the cell To further increase the sensitivity of the analysis protein complexes were eluted from the affinity matrix through calcium chelation prior to digestion and MS analysis improving the signal to noise ratio and identification of significant interactions.…”