Unit

UNIT 29.9 Expression and Purification of Haemophilus influenzae Rhomboid Intramembrane Protease GlpG for Structural Studies

  1. Pankaj Panwar,
  2. M. Joanne Lemieux

Published Online: 1 APR 2014

DOI: 10.1002/0471140864.ps2909s76

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Panwar, P. and Lemieux, M. J. 2014. Expression and Purification of Haemophilus influenzae Rhomboid Intramembrane Protease GlpG for Structural Studies. Current Protocols in Protein Science. 76:29.9:29.9.1–29.9.25.

Author Information

  1. Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada

Publication History

  1. Published Online: 1 APR 2014

Abstract

Rhomboid proteases are membrane-embedded proteases that cleave peptide bonds of transmembrane proteins. They play a variety of roles in cell signaling events. The rhomboid protease GlpG from Haemophilus influenzae (hiGlpG) is a canonical form of rhomboid protease having six transmembrane segments. In this unit, detailed protocols are presented for optimization of hiGlpG expression using the araBAD promotor system in the pBAD vector. The parameters for optimization include concentration of inducing agent, induction temperature, and time. Optimization of these key factors led to the development of a protocol yielding 1.6 to 2.5 mg/liter protein purified after ion metal affinity chromatography (IMAC). Further purification can include size exclusion chromatography (SEC). Curr. Protoc. Protein Sci. 76:29.9.1-29.9.25 © 2014 by John Wiley & Sons, Inc.

Keywords:

  • rhomboid protease;
  • expression study;
  • pBAD;
  • IMAC;
  • SEC