Design, Expression, and Purification of a Soluble Form of the Retina-Specific Membrane Transporter, ABCA4

The ATP-binding cassette transporter A-subfamily member, ABCA4, is highly expressed in rod and cone photoreceptors in the retina, where it transports cis- and trans-retinal and is indispensable for vision. Genetic mutations in the ABCA4 gene lead to a wide range of inherited retinal degenerative diseases, including Stargardt disease (STGD1) and autosomal recessive cone-rod dystrophy. It is an integral membrane protein with twelve transmembrane -helices that complicates studies with the full-length ABCA4 transporter. We have engineered the full-length ABCA4 by transforming its membrane helices, creating a soluble homolog (ABCA4s). Most hydrophobic residues in the membrane helices were substituted with structurally compatible but hydrophilic residues. The re-engineered ABCA4s was expressed in insect cells, and it was found in the cytosolic extract, which was purified by immunoaffinity chromatography. Purified ABCA4s was enzymatically active, all-trans-retinal stimulated its ATPase activity, and its activity remained stable. SignificanceABCA4 is a 12-pass transmembrane protein that plays essential roles in the human retina and multiple visual diseases. Historically, the purification of ABCA4 and other large membrane proteins has relied on detergent-based purification, which renders the proteins enzymatic activity highly unstable. We describe here the design of a truly soluble analog of ABCA4 with stable enzymatic activities. Its 12 transmembrane helices were transformed using selective amino acid substitution, and deleterious substitutions were carefully avoided. This soluble form will pave the way for mechanistic studies of the enzyme and its disease-causing genetic variants. The methodology described here should be widely applicable to other complex membrane proteins facilitating their studies without the need for reconstitution in lipids.