Abstract
A chymotrypsin inhibitor 2 variant, CI2(G83D), was expressed as a fusion with fd minor coat protein, pili, via a flexible linker (Gly-Ala 3) and displayed on the surface of bacteriophage. Biopanning with immobilised subtilisin BPN' demonstrated that the inhibitor-displaying phage could be selectively enriched 13-fold over control phage in one round of affinity purification. The inhibitor (incorporating the linker sequence) was also expressed as soluble protein in E. coli and purified to homogeneity. Kinetic analysis revealed the displayed inhibitor possesses inhibition characteristics that approximate those of this soluble inhibitor. The results demonstrate that functionally active inhibitor can be displayed on the phage surface. Further use of this technology should facilitate the selection of remodelled inhibitor variants exhibiting novel protease binding specificities.
Protein & Peptide Letters
Title:Bacteriophage Display of Chymotrypsin Inhibitor 2
Volume: 3 Issue: 6
Author(s): David l .R. Holmes, Dimitris Mantafounis, Walter H.J. Ward and Robin J. Leatherbarrow*
Affiliation:
- Department of Chemistry , Imperial College of Science, Technology and Medicine, London, SW7 2AZ, U.K
Abstract: A chymotrypsin inhibitor 2 variant, CI2(G83D), was expressed as a fusion with fd minor coat protein, pili, via a flexible linker (Gly-Ala 3) and displayed on the surface of bacteriophage. Biopanning with immobilised subtilisin BPN' demonstrated that the inhibitor-displaying phage could be selectively enriched 13-fold over control phage in one round of affinity purification. The inhibitor (incorporating the linker sequence) was also expressed as soluble protein in E. coli and purified to homogeneity. Kinetic analysis revealed the displayed inhibitor possesses inhibition characteristics that approximate those of this soluble inhibitor. The results demonstrate that functionally active inhibitor can be displayed on the phage surface. Further use of this technology should facilitate the selection of remodelled inhibitor variants exhibiting novel protease binding specificities.
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Cite this article as:
Holmes l .R. David, Mantafounis Dimitris, Ward H.J. Walter and Leatherbarrow J. Robin*, Bacteriophage Display of Chymotrypsin Inhibitor 2, Protein & Peptide Letters 1996; 3 (6) . https://dx.doi.org/10.2174/092986650306221101125251
DOI https://dx.doi.org/10.2174/092986650306221101125251 |
Print ISSN 0929-8665 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5305 |
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