HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hamilton, A. C. Articles by Ferrer, M. Search for Related Content PubMed PubMed Citation Articles by Hamilton, A. C. Articles by Ferrer, M. Social Bookmarking                   What's this?

A PDZ domain-based assay for measuring HIV protease activity: Assay design considerations

Department of Automated Biotechnology, Merck Research Laboratories, North Wales, Pennsylvania 19454, USA

Reprint requests to: Marc Ferrer, Department of Automated Biotechnology, Merck Research Laboratories, 502 Louise Lane, North Wales, PA 19454, USA; e-mail: marc_ferreralegre{at}merck.com; fax (267) 305-3625.

We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain•peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ-binding motif is exposed, and the product sequence bound specifically by a Eu³⁺chelate-labeled GST–PDZ ([Eu³⁺]GST–PDZ). The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ domain•peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ-based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD-95 was used, which preferentially recognizes the consensus sequence Ser-X-Val-COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.

Keywords: PDZ domains; TRET; ALPHA; HIV protease

Abbreviations: ALPHA, Amplified Luminescence Proximity Homogeneous Assay • CFTR, cystic fibrosis transmembrane regulator • GST-PDZ, Glutathione-S-Transferase fused to PDZ domain • HIV-PR, human immunodeficiency virus protease • mAb, monoclonal antibody • PBS, phosphate buffered saline • PBST, PBS with tween-20 • PBSTB, PBS with tween-20 and BSA • NHERF, Na+/H+ exchanger regulatory factor • PDZ, PSD95/Discs-large/ZO-1 • PSD95, postsynaptic density 95 • SA, streptavidin • TRET, time resolved energy transfer • [XL665]SA, allophycocyanin-conjugated streptavidin • , hydrophobic residue • , aromatic residue

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Ferrer, J. Maiolo, P. Kratz, J. L. Jackowski, D. J. Murphy, S. Delagrave, and J. Inglese Directed evolution of PDZ variants to generate high-affinity detection reagents Protein Eng. Des. Sel., April 1, 2005; 18(4): 165 - 173. [Abstract] [Full Text] [PDF]