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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
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Split green fluorescent protein as a modular binding partner for protein crystallization.

Hau B Nguyen1, Li-Wei Hung, Todd O Yeates

  • 1Bioscience Division, Los Alamos National Laboratory, MS M888, Los Alamos, NM 87545, USA.

Acta Crystallographica. Section D, Biological Crystallography
|December 7, 2013
PubMed
Summary
This summary is machine-generated.

This study introduces a novel protein crystallization method using split green fluorescent protein (GFP). This technique enhances protein structure determination by improving the ordered assembly of target proteins with GFP fragments.

Keywords:
crystallization reagentsgreen fluorescent proteinprotein crystallizationprotein expressionprotein taggingprotein-fragment complementationsplit GFPsplit proteinsynthetic symmetrization

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Area of Science:

  • Structural biology
  • Biochemistry
  • Protein engineering

Background:

  • Protein crystallization is crucial for determining protein structures.
  • Traditional methods like terminal fusion can be inefficient for crystallization.
  • Split protein systems offer modularity for protein complex formation.

Purpose of the Study:

  • To demonstrate a modular strategy for protein crystallization using split green fluorescent protein (GFP).
  • To improve the efficiency and structural order of protein-GFP complexes for crystallization.
  • To develop a versatile platform for generating protein crystallization variants.

Main Methods:

  • Insertion of a hairpin containing GFP beta-strands 10 and 11 into a target protein's surface loop.
  • Utilizing split GFP (GFP(1-9) and GFP(10-11)) for protein complex reconstitution.
  • Determining the crystal structure of the sfCherry-GFP(10-11) hairpin complex with GFP(1-9) at 2.6 Å resolution.

Main Results:

  • The hairpin insertion strategy creates two chain crossings, enhancing structural linkage between the target protein and reconstituted GFP.
  • The crystal structure confirmed a structurally ordered attachment of reconstituted GFP to the target protein (sfCherry).
  • This method provides a more robust connection compared to terminal GFP fusions.

Conclusions:

  • The demonstrated modular strategy enables efficient protein crystallization by utilizing split GFP.
  • This approach facilitates the rapid generation of diverse crystallization variants by combining target proteins with engineered GFP fragments.
  • This technique advances protein structure determination by offering a flexible and effective crystallization partner.