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Phage Display of Functional Human TNF- Converting Enzyme Catalytic Domain: A Rapid Method for the Production of Stabilized Proteolytic Proteins for Assay Development and High-Throughput Screening

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

Katrina Diener

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

Indravadan R. Patel

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

John K. Kawooya

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

Gary A. Martin

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

Preeti Yamdagni

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

Xin Zhang

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

Anthony Sandrasagra

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

Sudhir Sahasrabudhe

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

Steven J. Busch

Department of Functional Genomics, Aventis Pharmaceuticals, Inc., Bridgewater, NJ.

The catalytic domain of human tumor necrosis factor- (TNF-) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF- to generate the mature TNF- in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF--specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT. Both PDT and BET showed a similar specific cleavage profile against the defined substrates. Activity of the BET, however, was stable at 4 °C for less than 24 h. In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 °C. On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity. Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS.

Journal of Biomolecular Screening, Vol. 7, No. 5, 433-440 (2002)
DOI: 10.1177/108705702237675


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