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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Ludwig, Stephan / Sies, Helmut / Turk, Boris / Wittinghofer, Alfred

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Applicability of superfolder YFP bimolecular fluorescence complementation in vitro

Corinna Ottmann1 / Michael Weyand2 / Alexander Wolf3 / Jürgen Kuhlmann4a / Christian Ottmann5

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

2Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, D-44227 Dortmund, Germany

3Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, D-44227 Dortmund, Germany

4Department of Structural Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany

5Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, D-44227 Dortmund, Germany

Corresponding author

Citation Information: Biological Chemistry. Volume 390, Issue 1, Pages 81–90, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: 10.1515/BC.2009.008, November 2008

Publication History

Received:
2008-09-04
Accepted:
2008-10-01
Published Online:
2008-11-13

Abstract

Bimolecular fluorescence complementation (BiFC) using yellow fluorescent protein (YFP) is a widely employed method to study protein-protein interactions in cells. As yet, this technique has not been used in vitro. To evaluate a possible application of BiFC in vitro, we constructed a ‘superfolder split YFP’ system where 15 mutations enhance expression of the fusion proteins in Escherichia coli and enable a native purification due to improved solubility. Here, we present the crystal structure of ‘superfolder YFP’, providing the structural basis for the enhanced folding and stability characteristics. Complementation between the two non-fluorescent YFP fragments fused to HRas and Raf1RBD or to 14-3-3 and PMA2-CT52 resulted in the constitution of the functional fluorophore. The in vivo BiFC with these protein interaction pairs was demonstrated in eukaryotic cell lines as well. Here, we present for the first time BiFC in vitro studies with natively purified superfolder YFP fusion proteins and show the potential and drawbacks of this method for analyzing protein-protein interactions.

Keywords: 14-3-3; BiFC; fusicoccin; protein-protein interactions; Raf; Ras

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