Affinity Purification of Membrane Proteins.

Biochemical, biophysical, and structural studies of membrane proteins rely on the availability of highly pure and monodisperse membrane protein samples. One of the most powerful methods for isolation of the membrane protein of interest is affinity purification. This methodology typically relies on engineering an affinity tag into the protein of interest and an affinity resin that specifically recognizes the tag, allowing one to purify the target protein in a single step. In some cases, the affinity purification procedure is combined with additional steps to increase the purity and homogeneity of the final protein sample. Here, we describe several protocols for affinity purification of TSPO, a small membrane protein. The techniques we use include immobilized metal affinity chromatography (IMAC) and strep-II tag-based streptavidin affinity chromatography.

Characterisation of the ligand binding sites in the translocator protein TSPO using the chimeric bacterial-mammalian constructs.

The translocator protein TSPO is in an important diagnostic and therapeutic target in a range of pathologies, including neuroinflammation and cancer. Despite the availability of several structures of TSPO homologues, our understanding of the molecular determinants that govern high-affinity interactions of TSPO with its ligands is incomplete. Here, in order to decipher the key structural elements of TSPO responsible for interactions with its ligands, we designed a panel of chimeric proteins mimicking the mammalian substrate binding site grafted onto the backbone of the Rhodobacter sphaeroides TSPO homologue, RsTSPO. One of the designed chimeric constructs, RsMouse, could be heterologously expressed and displayed improved binding affinities for the known TSPO drugs diazepam, PK11195 and NBD-FGIN-1-27. Furthermore, the chimeric protein had improved interactions with NBD-cholesterol, a fluorescent analogue of the presumed natural substrate of TSPO. Partial modifications of the transmembrane helix bundle in the chimeric construct differentially affected binding of the TSPO drugs and the natural substrates of TSPO, consistent with the presence of multiple ligand binding sites in the protein. Based on the available structures of TSPO homologues, the substrate interactions may involve a lateral opening of the protein in the TM1-3, and stabilisation of TM4-5 is important for drug-like ligand binding. These observations are consistent with our experimental results, which show that the determinants of high-affinity ligand interactions of TSPO are distinct for different classes of ligands.

Expression and purification of the mammalian translocator protein for structural studies.

The translocator protein (TSPO) is an 18 kDa polytopic membrane protein of the outer mitochondrial membrane, abundantly present in the steroid-synthesising cells. TSPO has been linked to a number of disorders, and it is recognised as a promising drug target with a range of potential medical applications. Structural and biochemical characterisation of a mammalian TSPO requires expression and purification of the protein of high quality in sufficiently large quantities. Here we describe detailed procedures for heterologous expression and purification of mammalian TSPO in HEK293 cells. We demonstrate that the established procedures can be used for untagged TSPO as well as for C-terminally fused TSPO constructs. Our protocol can be routinely used to generate high-quality TSPO preparations for biochemical and structural studies.