Substrate Phage as a Tool to Identify Novel Substrate Sequences of Proteases

Authors: Ohkubo S.; Miyadera K.; Sugimoto Y.; Matsuo K-i.; Wierzba K.; Yamada Y.

Source: Combinatorial Chemistry & High Throughput Screening, Volume 4, Number 7, November 2001 , pp. 573-583(12)

Publisher: Bentham Science Publishers

Abstract:

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A “substrate phage library” has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.

Keywords: Substrate Phage; Proteases; membrane type-1 matrix metalloproteinase (MT1-MMP); Human Thrombin; granzyme B; Human MT1-MMP; Matrix metalloproteinase; Membrane type-1 matrix metalloproteinase; Tissue-type plasminogen activator; Urokinase-type plasminogen activator

Language: English

Document Type: Review article

DOI: http://dx.doi.org/10.2174/1386207013330788

Publication date: 2001-11-01

• Combinatorial Chemistry & High Throughput Screening publishes full length original research articles and reviews describing various topics in combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) and/or high throughput screening (e.g. developmental, practical or theoretical). Ancillary subjects of key importance, such as robotics and informatics, will also be covered by the journal. In these respective subject areas, Combinatorial Chemistry & High Throughput Screening is intended to function as the most comprehensive and up-to-date medium available. The journal should be of value to individuals engaged in the process of drug discoveryand development, in the settings of industry, academia or government.

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• By this author: Ohkubo S. ;  Miyadera K. ;  Sugimoto Y. ;  Matsuo K-i. ;  Wierzba K. ;  Yamada Y.

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