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Esther E. Biswas-Fiss, Stephanie Affet, Subhasis B. Biswas; Expression of the Human Retina Specific ABC Transporter, ABCA4, in Virus-Like Particles. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1610.
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Mutations in the ABCA4 gene cause a major proportion of autosomal recessive retinal degenerations (RD) with macular involvement, including Stargardt disease (STGD1), autosomal recessive retinitis pigmentosa (arRP) and cone-rod dystrophy (CRD. Despite the biological role of ABCA4 and its relevance to serious visual disorders, knowledge of the biochemical, structural, and functional properties of this membrane protein remains limited. The purpose of this study was to develop a platform for the analysis of the biochemical consequences of a given mutation on ABCA4 function.
Full length human ABCA4 cDNA was cloned into a mammalian expression vector under the control of a CMV promoter. The recombinant ABCA4 construct was co-transfected into HEK293T cell line harboring a plasmid expressing the structural gag and pol proteins of murine leukemia virus (MLV). MLV gag and pol proteins create virus-like particles (virions without viral RNA), which are enriched with recombinant ABCA4 protein. Verification of expression was carried out through analysis of isolated virus-like particles via Western Blotting using ABCA4 domain specific antibodies. The isolated ABCA4 expressing virus-like particles were used for further structure function analysis.
Analysis of the virus-like particle fraction harboring the ABCA4 plasmids demonstrated the expression of a 230 kDa polypeptide that cross-reacted with domain specific ABCA4 antibodies. No expression was observed in cells harboring the control plasmid. The wild-type ABCA4 protein in isolated VLPs was biologically active with respect to the ability to hydrolyze ATP and appeared to display a uniform membrane topology.
We have successfully expressed the ABCA4 protein in virus-like particles. ABCA4-VLPs represent a useful approach for the analysis of ABCA4-mediated retinal transport and effects of genetic mutations on the transport process.
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