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Protease Substrate Screening

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Phage display peptide libraries that display random 5-20 mer peptides have been widely used to determine protease substrate (sequence) specificity for a large number of enzymes and semi-purified cell extracts. This technique has been playing a critical role in designing inhibitors and activators of proteases.

Protease Substrate Screening

In comparison with phage display peptide libraries used in other fields, phage display peptide libraries used to identify the preferred substrate sequences of a protease usually express a fusion tag at the N-terminus of the peptide sequences, while, like in regular phage display peptide libraries, the minor coat viral protein pIII is located at the C-terminus of the peptide sequences. The N-terminal fusion tag is intended to bind the phage particles to a solid support. Scientists from Creative Biolabs have designed and built up a unique tag, HiAffi-tag, which is derived from Protein G, thus has strong binding affinity to most IgG coated on a solid surface. In contrast to His Tag and Strepavidin Binding Motifs used in such libraries, HiAffi-tag enables the best capture of the phage particles to the solid support and the least steric hindrance for proteolysis of the substrate peptides.

During library screening against a particular protease, phage particles that express sensitive peptide sequences are cleaved off the solid support. After that, the free phages are amplified, immobilized and subjected to protease cleavage again. After several rounds of screening, selected phage clones are DNA sequenced and common features of the peptide sequences are determined. These peptide sequences are good candidates for further identification of the substrates of the protease.

Creative Biolabs also developed proprietary in silicon screening and computer aided rational design methods to use such peptide sequences, which must fit into the active site of the protease, as leads to develop inhibitors and activators of a protease.




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