A robust purification strategy to accelerate membrane proteomics.
The preparation of large quantities of purified membrane proteins for structural studies presents significant difficulties. Central among these are the frequent toxicity associated with over-expressing membrane targets and the difficulty associated with identifying the appropriate detergents for their solubilization and purification. To begin addressing these challenges, and lay the groundwork for membrane structural genomics efforts, we have developed a robust strategy for the expression and purification of large numbers of prokaryotic membrane proteins. Our approach rapidly identifies highly expressed targets and greatly simplifies their solubilization and purification. In this review, specific, hands-on protocols are provided for the expression and purification of CorA magnesium transporters. These methods form the basis for the expression and purification of many other membrane proteins, as discussed.
Dobrovetsky
Elena
E
Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, ON, Canada M5G 1L6.
Menendez
Javier
J
Edwards
Aled M
AM
Koth
Christopher M
CM
D003001:Cloning, Molecular / Q000379:methods; D003460:Crystallization; D003902:Detergents; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*
D003902:Detergents; D008565:Membrane Proteins