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Esben Meldgaard Høgh Quistgaard

High-throughput analytical gel filtration screening of integral membrane proteins for structural studies

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Christian Löw, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. christian.low@ki.se
  • ,
  • Per Moberg
  • ,
  • Esben M Quistgaard
  • Marie Hedrén
  • ,
  • Fatma Guettou
  • ,
  • Jens Frauenfeld
  • ,
  • Lars Haneskog
  • ,
  • Pär Nordlund

BACKGROUND: Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning.

METHODS: Here we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC).

RESULTS: To validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy.

CONCLUSIONS: We have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up.

GENERAL SIGNIFICANCE: The screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins.

Original languageEnglish
JournalB B A - General Subjects
Volume1830
Issue6
Pages (from-to)3497-508
Number of pages12
ISSN0006-3002
DOIs
Publication statusPublished - Jun 2013
Externally publishedYes

    Research areas

  • Chromatography, Gel, Escherichia coli, Escherichia coli Proteins, Membrane Proteins, Journal Article, Research Support, Non-U.S. Gov't

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