Unit

UNIT 29.6 High-Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

  1. Renato Bruni,
  2. Brian Kloss

Published Online: 5 NOV 2013

DOI: 10.1002/0471140864.ps2906s74

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Bruni, R. and Kloss, B. 2013. High-Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli. Current Protocols in Protein Science. 74:29.6:29.6.1–29.6.34.

Author Information

  1. New York Consortium on Membrane Protein Structure (NYCOMPS), New York Structural Biology Center (NYSBC), New York

Publication History

  1. Published Online: 5 NOV 2013

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. Curr. Protoc. Protein Sci. 74:29.6.1-29.6.34. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • Escherichia coli;
  • high-throughput;
  • cloning/expression;
  • recombinant protein expression;
  • membrane protein;
  • ligation-independent cloning (LIC);
  • membrane protein purification