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Related Experiment Video

Updated: Jun 28, 2026

Bimolecular Fluorescence Complementation
08:54

Bimolecular Fluorescence Complementation

Published on: April 15, 2011

Applicability of superfolder YFP bimolecular fluorescence complementation in vitro.

Corinna Ottmann1, Michael Weyand, Alexander Wolf

  • 1Department of Structural Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany.

Biological Chemistry
|November 15, 2008
PubMed
Summary

This study introduces a superfolder split yellow fluorescent protein (YFP) system for in vitro bimolecular fluorescence complementation (BiFC). This novel approach enables protein-protein interaction analysis outside of living cells.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Bimolecular fluorescence complementation (BiFC) is a common technique for studying protein-protein interactions in living cells.
  • The application of BiFC in in vitro settings has been limited due to challenges with protein expression and purification.

Purpose of the Study:

  • To develop and validate a superfolder split yellow fluorescent protein (YFP) system for in vitro BiFC.
  • To investigate the structural basis for the enhanced folding and stability of the superfolder YFP.
  • To assess the potential and limitations of in vitro BiFC using purified proteins.

Main Methods:

  • Construction of a superfolder split YFP system with 15 mutations for enhanced expression and solubility in E. coli.
  • Native purification of superfolder YFP fusion proteins.

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Last Updated: Jun 28, 2026

Bimolecular Fluorescence Complementation
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Published on: April 15, 2011

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  • Determination of the crystal structure of superfolder YFP.
  • Demonstration of in vitro BiFC with specific protein interaction pairs (HRas/Raf1RBD, 14-3-3/PMA2-CT52).
  • Validation of in vivo BiFC in eukaryotic cell lines.

    • Main Results:

    • The superfolder split YFP system demonstrated enhanced expression and solubility in E. coli.
    • The crystal structure of superfolder YFP revealed structural features contributing to its improved folding and stability.
    • Successful in vitro complementation and fluorescence reconstitution were achieved with purified superfolder YFP fusion proteins.
    • In vivo BiFC experiments confirmed the functionality of the system in eukaryotic cells.

    Conclusions:

    • The developed superfolder split YFP system enables the first successful in vitro BiFC analysis of protein-protein interactions using natively purified proteins.
    • The structural insights provide a basis for understanding the enhanced properties of superfolder YFP.
    • This method offers a valuable tool for studying protein interactions in vitro, with identified potential and limitations.