PEDS Advance Access originally published online on April 8, 2005
Protein Engineering Design and Selection 2005 18(4):165-173; doi:10.1093/protein/gzi018
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Directed evolution of PDZ variants to generate high-affinity detection reagents
1Department of Automated Biotechnology, Merck and Co., Inc., 502–503 Louise Lane, North Wales, PA 19454, 2BioTech Studio LLC, 3701 Market Street, Philadelphia, PA 19104 and 3Fraunhofer USA, Center for Molecular Biotechnology, 9 Innovation Way, Newark, DE 19711, USA
4 To whom correspondence should be addressed, at BioTech Studio LLC
5 To whom correspondence should be addressed. Present address: NIH Chemical Genomics Center, 9800 Medical Center Drive, MSC: 3370, Bethesda, MD 20892-3370, USA. E-mail: sdelagrave{at}biotechstudio.com or jinglese{at}mail.nih.gov
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 ‘ß1–ß3’ 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.
Keywords: directed evolution/high-affinity detection reagents/PDZ variants
Received October 3, 2004; revised January 17, 2005; accepted January 18, 2005.
Edited by William DeGrado
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