Expression, Purification and Characterization of Bacterial and Human Translocator Protein 18 kDa (TSPO)

Abstract

The Translocator Protein 18 kDa (TSPO) has been identified as a key player in cholesterol and porphyrin transport, apoptotic signaling, cancer development as well as neurological disease. Despite extensive research, the molecular basis of the interactions of ligands with TSPO, including benzodiazepine drugs, cholesterol and porphyrins remains unclear. We successfully expressed and purified the recombinant human TSPO (HsTSPO) and its close homolog, TSPO from Rhodobacter sphaeroides (RsTSPO) in E. coli and characterized their biophysical and biochemical properties. Both human and Rhodobacter TSPO were purified to homogeneity by Ni-NTA affinity followed by size exclusion chromatography. The protein-detergent complex of HsTSPO behaves as a hexamer of ∼160 kDa and RsTSPO as a dimer of ∼66 kDa as measured by size exclusion. The binding of purified HsTSPO and RsTSPO with endogenous ligands protoporphyrin IX (PpIX), cholesterol, and the drug ligand PK11195, were measured using a tryptophan fluorescence quenching assay. PpIX showed μM affinity to both HsTSPO and RsTSPO. The binding of PK11195 to HsTSPO showed bi-phasic behavior with apparent Kds in the nM range, while RsTSPO showed similar bi-phasic behavior in the μM range. Preliminary data suggests nM affinity of cholesterol for HsTSPO but μM affinity for RsTSPO. Knowledge of the biophysical and biochemical properties of TSPO obtained in this study confirms the results of some previous studies and provides important information for ongoing crystallographic efforts to determine structure. (Supported by the Center for Mitochondrial Science and Medicine, Strategic Partnership Grant, Michigan State University Foundation).