Format

Send to:

Choose Destination
See comment in PubMed Commons below
Nat Protoc. 2012 Feb 9;7(3):453-66. doi: 10.1038/nprot.2011.453.

Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells.

Author information

  • 1Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA.

Abstract

X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 (HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI(-)), and was recently used in our 2.1-Å X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of ∼0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8-10 weeks.

PMID:
22322218
[PubMed - indexed for MEDLINE]
PMCID:
PMC3613139
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for Nature Publishing Group Icon for PubMed Central
    Loading ...
    Write to the Help Desk