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PEDS Advance Access published online on March 21, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzl004
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received February 10, 2006
Accepted February 14, 2006

Article

Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries

Patrick Amstutz 1 4, Holger Koch 2 4, H. Kaspar Binz 2, Stefan A. Deuber 3, and Andreas Plückthun 2 *

1 Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; Present address: Molecular Partners AG, c/o Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
2 Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
3 Institut für Medizinische Virologie der Universität Zürich, Gloriastrasse 30, Zürich, Switzerland

* To whom correspondence should be addressed.
Andreas Plückthun, E-mail: plueckthun{at}bioc.unizh.ch


  Abstract

We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 105 per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cells.

Keywords: ribosome display; protein fragment complementation assay; MAP kinase; designed ankyrin repeat proteins (DARPins); intrabody.
4These authors contributed equally to this work.
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