Overexpression and Purification of Mouse Divalent Metal Transporter 1 (mDMT1) from Saccharomyces cerevisiae

Abstract: The membrane integrated divalent metal transporter 1 (DMT1) functions as a transporter of mainly iron across cellular membranes within the human body. Studies indicate that dysregulated transport by DMT1 causes accumulation of iron within the midbrain leading to neuronal cell death – a common symptom among patients suffering from Parkinson’s disease (PD). Detailed structural information of DMT1 is currently lacking but may aid in drug development for PD. To solve the 3D crystal structure of DMT1, high-purity mono disperse protein must first be obtained. To this end, we expressed mouse DMT1 (mDMT1) in Saccharomyces cerevisiae and developed protocols to purify this protein. Based on a detergent screen and our purification trials we find that mDMT1 is soluble in n-dodecyl-β-D-maltpyranoside (DDM) and an addition of cholesteryl hemisuccinate (CHS) increases solubility of the protein in solution. Furthermore, we determine that the highest protein concentration by S. cerevisiae is obtained at 23 °C after 43 h. Although none of the performed purification trials was entirely successful at yielding mDMT1 samples suitable for crystallization, we present guidelines as to what areas of the protocol may be optimized to achieve this goal.