Ratiocinative screen of eukaryotic integral membrane protein expression and solubilization for structure determination

  • Franklin A. Hays
  • Zygy Roe-Zurz
  • Min Li
  • Libusha Kelly
  • Franz Gruswitz
  • Andrej Sali
  • Robert M. Stroud
Article

DOI: 10.1007/s10969-008-9046-7

Cite this article as:
Hays, F.A., Roe-Zurz, Z., Li, M. et al. J Struct Funct Genomics (2009) 10: 9. doi:10.1007/s10969-008-9046-7

Abstract

Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering high-quality, responsive non-redundant targets for crystallization. This “discovery-oriented” pipeline sidesteps two significant bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column—a hallmark of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker’s yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane proteomes.

Keywords

Discovery-oriented screenMembrane protein structureStructural genomicsEukaryotic integral membrane proteinSaccharomyces cerevisiae

Abbreviations

DDM

n-Dodecyl-β-d-maltopyranoside

IMAC

Immobilized metal affinity chromatography

SEC

Size exclusion chromatography

TMH

Transmembrane helices

LIC

Ligase independent cloning

OG

n-Octyl-β-d-glucoside

FC-12

n-Dodecylphosphocholine

IMP

Integral membrane protein

Supplementary material

10969_2008_9046_MOESM1_ESM.xls (38 kb)
MOESM1 (XLS 38 kb)

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Franklin A. Hays
    • 1
  • Zygy Roe-Zurz
    • 2
  • Min Li
    • 2
  • Libusha Kelly
    • 3
    • 4
    • 5
    • 6
  • Franz Gruswitz
    • 2
  • Andrej Sali
    • 4
    • 5
    • 6
    • 7
  • Robert M. Stroud
    • 1
  1. 1.Department of Biochemistry and BiophysicsUniversity of California at San FranciscoSan FranciscoUSA
  2. 2.Membrane Protein Expression CenterUniversity of California at San FranciscoSan FranciscoUSA
  3. 3.Graduate Group in BioinformaticsUniversity of California at San FranciscoSan FranciscoUSA
  4. 4.Center for the Structure of Membrane ProteinsUniversity of California at San FranciscoSan FranciscoUSA
  5. 5.Department of Pharmaceutical ChemistryUniversity of California at San FranciscoSan FranciscoUSA
  6. 6.California Institute for Quantitative BiosciencesUniversity of California at San FranciscoSan FranciscoUSA
  7. 7.Department of Bioengineering and Therapeutic SciencesUniversity of California at San FranciscoSan FranciscoUSA