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METHODS & TECHNIQUES
Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry
Michael Brauchle, Simon Hansen, Emmanuel Caussinus, Anna Lenard, Amanda Ochoa-Espinosa, Oliver Scholz, Simon G. Sprecher, Andreas Plückthun, Markus Affolter
Biology Open 2014 3: 1252-1261; doi: 10.1242/bio.201410041
Michael Brauchle
1Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
2Department of Zoology, University of Fribourg, Chemi du Musée 10, 1700 Fribourg, Switzerland
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Simon Hansen
3Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
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Emmanuel Caussinus
1Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
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Anna Lenard
1Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
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Amanda Ochoa-Espinosa
1Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
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Oliver Scholz
3Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
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Simon G. Sprecher
2Department of Zoology, University of Fribourg, Chemi du Musée 10, 1700 Fribourg, Switzerland
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Andreas Plückthun
3Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
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  • For correspondence: plueckthun@bioc.uzh.ch Markus.Affolter@unibas.ch
Markus Affolter
1Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
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  • For correspondence: plueckthun@bioc.uzh.ch Markus.Affolter@unibas.ch
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ABSTRACT

Protein–protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.

Footnotes

  • ↵* These authors contributed equally to this work

  • ↵‡ These authors contributed equally to this work

  • Author Contributions M.B., S.H., A.P. and M.A. conceived and designed the experiments. M.B., S.H., E.C., A.L., A.O.-E., O.S. and S.G.S. performed the experiments and analysed the data. M.B., S.H., A.P. and M.A. wrote paper.

  • Competing interests The authors have no competing interests to declare. A.P. is a cofounder and shareholder of Molecular Partners AG, commercializing the DARPin technology.

  • Received August 26, 2014.
  • Accepted October 11, 2014.
  • © 2014. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Keywords

  • DARPin
  • GFP
  • mCherry
  • Protein interference

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METHODS & TECHNIQUES
Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry
Michael Brauchle, Simon Hansen, Emmanuel Caussinus, Anna Lenard, Amanda Ochoa-Espinosa, Oliver Scholz, Simon G. Sprecher, Andreas Plückthun, Markus Affolter
Biology Open 2014 3: 1252-1261; doi: 10.1242/bio.201410041
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METHODS & TECHNIQUES
Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry
Michael Brauchle, Simon Hansen, Emmanuel Caussinus, Anna Lenard, Amanda Ochoa-Espinosa, Oliver Scholz, Simon G. Sprecher, Andreas Plückthun, Markus Affolter
Biology Open 2014 3: 1252-1261; doi: 10.1242/bio.201410041

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