Isolating substrates for an engineered α-lytic protease by phage display
Date
2003
Authors
Lien, S.
Francis, G.
Graham, L.
Wallace, J.
Editors
Advisors
Journal Title
Journal ISSN
Volume Title
Type:
Journal article
Citation
The Protein Journal, 2003; 22(2):155-166
Statement of Responsibility
Samantha Lien, Geoffrey L. Francis, Lloyd D. Graham, and John C. Wallace
Conference Name
Abstract
Panning of a substrate phage library with an α-lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered α-lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P₄ positions, whereas Asp dominated the putative P₁ position.
School/Discipline
Dissertation Note
Provenance
Description
The original publication is available at www.springerlink.com
Access Status
Rights
© 2003 Plenum Publishers Corporation