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E.E. Biswas; Heterologous Expression Of The Human Retina Specific ABC Transporter, ABCR, In The Yeast Saccharomyces Cerevisiae. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1250.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Human genetic studies have linked mutations in the ABCR gene to a number of inherited macular degenerations. Analysis of ABCR knock out mice suggests that the protein acts as a flipase of N–retinylidine–PE. Presumably, disease–associated mutations in ABCR lead to defects in the transport function. Our goal is to develop an in vitro system in order that this hypothesis could be tested. The purpose of this study is to express the ABCR protein in a heterologous membrane system with a goal of developing an in vitro transport system for analyzing the transport function. Methods: Full–length ABCR cDNA and certain genetic mutants were cloned into Saccharomyces cerevisiae pYES expression plasmid containing an ADH1 promoter for constitutive expression. Yeast strains were transformed with recombinant and control plasmids. Cells were grown at 30°C to mid log phase and time course analysis of expression was carried out to optimize expression. Verification of expression was carried out through solubilization of isolated yeast membrane using 0.5% SDS, followed by Western Blotting using RIM3F4 peptide specific antibody. Recombinant ABCR proteins were purified using an immunoaffinity method. The recombinant ABCR proteins were reconstituted using brain polar lipid for further structure function analysis. Results: Analysis of the yeast membrane fraction harboring the pYES–ABCR plasmids demonstrated the expression of a ∼230 kDa polypeptide that cross–reacted with the Rim3F4 antibody. No expression was observed in cells harboring the control pYES plasmid. The purified wild–type protein, reconstituted in brain polar lipids, was biologically active with respect to the ability to hydrolyze ATP. Conclusion: We have successfully expressed the ABCR protein in this heterologous system. Current studies are underway in which the recombinant ABCR proteins will be used for the analysis of retinal transport and effects of genetic mutations on the transport process.
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