Combinatorial method for overexpression of membrane proteins in Escherichia coli

Shani Leviatan, Keisuke Sawada, Yoshinori Moriyama, Nathan Nelson

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.

Original languageEnglish
Pages (from-to)23548-23556
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number31
DOIs
Publication statusPublished - Jul 30 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Combinatorial method for overexpression of membrane proteins in Escherichia coli'. Together they form a unique fingerprint.

  • Cite this