High‐Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

Recently, several structural genomics centers have been established and a remarkable number of three‐dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over‐expression and purification of membrane proteins is a much more arduous process. By using high‐throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression, and screening of membrane proteins using high‐throughput methodologies developed in the laboratory. Basic Protocol 1 describes cloning of inserts into expression vectors by ligation‐independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that do express on the miniscale, Basic Protocols 3 and 4 outline the methods employed for the expression and purification of targets on a midi‐scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. Curr. Protoc. Protein Sci . 74:29.6.1‐29.6.34. © 2013 by John Wiley & Sons, Inc.