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

Abstract

The catalytic domain of human tumor necrosis factor-α (TNF-α) 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-α to generate the mature TNF-α 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-α-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.