Human Plasma Membrane Anion Exchanger, AE1: Over‐expression in Saccharomyces cerevisiae

Abstract

Anion exchanger 1 (AE1) facilitates the exchange of chloride for bicarbonate across the plasma membrane of renal and erythrocyte membranes. The 55 kDa membranedomain of AE1 alone is responsible for its transport function, however a high resolution structure of this domain has not been solved. The limiting factor to obtaining acrystal structure is the availability of homogenous, purified AE1. Here we developed a novel expression and efficient purification system for AE1, using S. cerevisiaeto allow for large‐scale expression, while maintaining proper folding of AE1. The expression construct consists of the AE1 membrane domain (AE1MD), residues 388–911 of human AE1, followed by the 9 C‐terminal amino acids of rhodopsin. Expression was under the control of the constitutive promoter and transcriptionalterminator of the yeast H+‐ATPase gene. AE1MD was expressed in S. cerevisiae, strain BJ5457, to 0.3 mg AE1MD per litre of culture. We characterized the cellularlocalization of AE1MD by confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation. AE1MD structure was assessed by binding to animmobilized anion exchange inhibitor (SITS‐Affi‐gel). Once solubilized by the detergent n‐dodecyl‐β‐D‐maltoside, AE1MD was purified with a 1D4 antibody affinitycolumn, which recognizes the rhodopsin tag on AE1MD. This research is supported by the Canadian Institutes of Health Research.