Purification of functional human Cl(-)/HCO(3)(-) exchanger, AE1, over-expressed in Saccharomyces cerevisiae.
There is no high-resolution structure for the membrane domain of the human erythrocyte anion exchanger, AE1 (Band 3). In this report, we have developed an expression and purification strategy for AE1 to be used in crystallization trials. Saccharomyces cerevisiae strain BJ5457 was transformed with an expression vector encoding the AE1 membrane domain (AE1MD, amino acids 388-911), fused C-terminally to an epitope tag, corresponding to the nine C-terminal amino acids of rhodopsin. The fusion protein, AE1MD-Rho, was expressed at a concentration of 0.3 mg/l of culture. Confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation revealed that AE1MD-Rho did not process to the plasma membrane of S. cerevisiae, but was retained in an intracellular membrane fraction. Treatment with the endoglycosidase, PNGase F, showed that AE1MD-Rho is not N-glycosylated. AE1MD-Rho solubilized from yeast membranes, with Fos-choline detergent, was purified to 93% homogeneity in a single-step, using a 1D4 antibody affinity resin, in amounts up to 2.5 mg from 18 l of culture. The ability of purified AE1MD-Rho to transport sulfate was examined in reconstituted vesicles. The rate of sulfate efflux mediated by vesicles reconstituted with AE1MD-Rho was indistinguishable from vesicles with purified erythrocyte-source AE1. Using this purification strategy, sufficient amounts of functional, homogeneous AE1MD-Rho can be purified to enable crystallization trials.
Bonar
Pamela
P
Membrane Protein Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Canada T6G 2H7.
Casey
Joseph R
JR
D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000838:Anions / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003902:Detergents; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D012243:Rhodopsin / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure; D015854:Up-Regulation
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D003902:Detergents; D012243:Rhodopsin