The major bottleneck to the application of high-resolution techniques such as crystallographic X-ray diffraction and spectroscopic analyses to resolve the structure of mammalian membrane proteins has been the ectopic expression and purification of sufficient quantities of non-denatured proteins. This has been especially problematic for members of the major facilitator superfamily, which includes the family of mammalian glucose transporters. A simple and rapid method is described for the purification of milligram quantities of recombinant GLUT1 and GLUT4, two of the most intensively studied GLUT isoforms, after ectopic expression in Pichia pastoris. The proteins obtained were >95% pure and exhibited functional transport and ligand-binding activities. (C) 2009 Elsevier Inc. All rights reserved.