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https://hdl.handle.net/2440/3091
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Type: | Journal article |
Title: | Isolating substrates for an engineered α-lytic protease by phage display |
Other Titles: | Isolating substrates for an engineered alpha-lytic protease by phage display |
Author: | Lien, S. Francis, G. Graham, L. Wallace, J. |
Citation: | The Protein Journal, 2003; 22(2):155-166 |
Publisher: | Kluwer Academic/plenum Publ |
Issue Date: | 2003 |
ISSN: | 0277-8033 1573-4943 |
Statement of Responsibility: | Samantha Lien, Geoffrey L. Francis, Lloyd D. Graham, and John C. Wallace |
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. |
Keywords: | α-Lytic protease substrate phage display enzyme specificity |
Description: | The original publication is available at www.springerlink.com |
Rights: | © 2003 Plenum Publishers Corporation |
DOI: | 10.1023/A:1023475030579 |
Published version: | http://dx.doi.org/10.1023/a:1023475030579 |
Appears in Collections: | Aurora harvest 2 Molecular and Biomedical Science publications |
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