TSPO is a 18 kDa membrane protein that exists in mammalian as two isoforms 1 and 2. They are involved in different functions and are located in different membranes. TSPO1 is mainly located in outer mitochondrial membrane, whereas TSPO2 is encountered in plasma membrane of red blood cells. Determination of their structures is a milestone to understand their function. Their natural abundance is not sufficient to get large amounts usually required for structural studies. We described heterologous overexpression in both bacterial and cell-free system and purification on immobilized-metal affinity chromatography (IMAC) of both proteins. Using the same vector, TSPO1 is mostly recovered in bacterial inclusion bodies whereas TSPO2 is found in both bacterial cytosol and inclusion bodies, but in low amounts. Cell-free expression was the best system to overexpress pure TSPO2.
Keywords: Cell-free; Escherichia coli bacteria; Expression vector; NMR; Ni-NTA; TSPO.