Membrane Protein Production using Retroviral Vehicles

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DESCRIPTION (provided by applicant): Despite their importance, proteins that span the membrane multiple times present a unique set of challenges for structural analyses such as x-ray crystallography. Large portions of these proteins are hydrophobic, the proteins are topologically complex, and removal from their lipid bilayer results in loss of their native structure. Because of their complex folding and trafficking, many membrane proteins, especially those of eukaryotic origin, are difficult to express. Expression limitations can sometimes be overcome, but the membrane proteins must still be purified away from the cell, a process that is limited by protein heterogeneity in lysates, unwanted membrane proteins, and available detergents. Without sufficient quantities of structurally intact membrane protein to start with, many of the downstream steps of structural analysis cannot be optimized or even initiated. Although it is not certain what advances need to be made to routinely obtain membrane protein crystal structures, it is clear that traditional materials and methods are not sufficient, especially for eukaryotic membrane proteins. The purpose of this proposal is to develop a system capable of producing multi-milligram quantities of structurally intact, homogeneous membrane protein that can be used for structural analysis.