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PEDS Advance Access published online on September 26, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi060
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received May 12, 2005
Revised August 11, 2005
Accepted August 16, 2005

Article

Directed evolution for the development of conformation-specific affinity reagents using yeast display

Jane M. Weaver-Feldhaus 1, Keith D. Miller 1, Michael J. Feldhaus 2, and Robert W. Siegel 1*

1 Pacific Northwest National Laboratory, Richland, WA 99352, USA
2 Merrimack Pharmaceuticals, 101 Binney Street, Cambridge, MA 02142, USA

* To whom correspondence should be addressed.
Robert W. Siegel, E-mail: robert.siegel{at}abbott.com


  Abstract

Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca2+ binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca2+-bound (Ca2+-CaM) or Ca2+-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (Kd = 0.8 nM) and specificity (>1000-fold) for Ca2+-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (Kd = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.

Keywords: calmodulin; directed evolution; flow cytometry; recombinant antibody; yeast display.
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