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Structural Studies of Human Plasma Membrane Anion Exchanger, AE1, Expressed in Saccharomyces cerevisiae
Author(s) -
Bonar Pamela Therese,
Casey Joseph R.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.800.5
Subject(s) - band 3 , chemistry , membrane , amino acid , biochemistry , affinity chromatography , membrane protein , chromatography , enzyme
Anion exchanger 1 (AE1), which is expressed in the renal and erythrocyte membranes, functions to exchange chloride for bicarbonate across the plasma membrane. While there is a high‐resolution structure of the N‐terminal cytoplasmic domain of AE1, there is no high‐resolution structure of the 55 kDa AE1 membrane domain. We have developed an expression and purification system for AE1, which takes advantage of S. cerevisiae as a large‐scale expression system of properly folded homogeneous AE1. The expression construct (AE1‐Rho) consists of the AE1 membrane domain, amino acids 388 to 911 of human AE1, followed by the 9 C‐terminal amino acids of rhodopsin. Milligram quantities of AE1‐Rho have been purified in the presence of the detergent fos‐choline, by a 1D4 antibody affinity column, which recognizes the 9 C‐terminal amino acids of rhodopsin tagged to the AE1 membrane domain. AE1‐Rho is non‐glycosylated and prodominantly dimeric, as determined by respectively endoglycosidase F treatment and size exclusion chromatography. AE1‐Rho secondary structure was assessed by circular dichroism. The functional activity of AE1‐Rho was measured by radioactive sulfate efflux assays in reconstituted proteoliposomes. This research is supported by the Canadian Institutes of Health Research.