title: High-throughput cloning and expression of integral membrane proteins in Escherichia coli.
abstract: 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.
authors: New York Consortium on Membrane Protein Structure (NYCOMPS), New York Structural Biology Center (NYSBC), New York.
authors: Renato
authors: R
authors: Bruni
authors: New York Consortium on Membrane Protein Structure (NYCOMPS), New York Structural Biology Center (NYSBC), New York.
authors: Brian
authors: B
authors: Kloss
mesh_terms: D002853:Chromatography, Liquid; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D057166:High-Throughput Screening Assays; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins
keywords: Escherichia coli; cloning/expression; high-throughput; ligation-independent cloning (LIC); membrane protein; membrane protein purification; recombinant protein expression