Biochemical and biophysical characterization of a prokaryotic Mg2+ ion channel: Implications for cost-effective purification of membrane proteins

Publication date: Available online 24 April 2019Source: Protein Expression and PurificationAuthor(s): Satyaki Chatterjee, Anindita Das, H. RaghuramanAbstractAlthough magnesium is the second most abundant cation present in the cell, the transport mechanism of Mg2+ across membranes is poorly understood. Importantly, the prokaryotic MgtE Mg2+ channel is related to mammalian SLC41A1 transporters and, therefore, biochemical and biophysical characterization of MgtE and its orthologs assumes significance. To date, the purification and structure determination of MgtE from Thermus thermophilus has been carried out using the widely used nonionic detergent, n-dodecyl-β-d-maltopyranoside (DDM). However, DDM is an expensive detergent and alternative methods to produce high-quality proteins in stable and functional form will be practically advantageous to carry out structural studies in a cost-effective manner. In this work, we have utilized ‘dual-detergent strategy’ to successfully purify MgtE channel in a stable and functional form by employing relatively inexpensive detergents (Triton X-100 and Anzergent 3–14) for membrane solubilization and subsequently changed to DDM during purification. Our results show that Triton X-100 and Anzergent 3–14 extract MgtE well and the quality of purified protein is comparable to DDM-extracted MgtE. Interestingly, addition of high concentration of salt and glycerol during solubilization does not significantly affect the quantity and quality of M...
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research