PEDS Advance Access published online on April 8, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi018
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1 Department of Automated Biotechnology, Merck and Co., Inc., 502-503 Louise Lane, North Wales, PA 19454, USA
* To whom correspondence should be addressed. High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific ‘
Received October 3, 2004
Revised January 17, 2005
Accepted January 18, 2005
Communication
Directed evolution of PDZ variants to generate high-affinity detection reagents
2 BioTech Studio LLC, 3701 Market Street, Philadelphia, PA 19104, USA; Fraunhofer USA, Center for Molecular Biotechnology, 9 Innovation Way, Newark, DE 19711, USA
3 Fraunhofer USA, Center for Molecular Biotechnology, 9 Innovation Way, Newark, DE 19711, USA
Simon Delagrave, E-mail: sdelagrave{at}biotechstudio.com
Abstract 
1-3’ interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.
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