Linked Life Data

20615196

Source:http://linkedlifedata.com/resource/pubmed/id/20615196

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
9
pubmed:dateCreated
2010-9-20
pubmed:abstractText
A recombinant Haematobia irritans irritans trypsin inhibitor (HiTI - Mw 7030 kDa)) phagemid library was constructed and displayed functionally on the tip of the filamentous M13 phage. A combinatorial library of 7.2 x 10(6) mutants was created with HiTI mutations restricted to the P1'-P3' and P5' positions of the reactive site. This combinatorial library was selected for trypsin-like Pr2 proteases of Metarhizium anisopliae fungus, and 11 HiTI mutants containing the following substitutions: K17G, S18R, D19G, S21A, among 60 sequenced clones, were obtained. In order to confirm the inhibitory activity of the selected sequences, we transferred the selected sequence to the shortest protease inhibitor, the sunflower trypsin inhibitor (SFTI), for inhibitory activity analysis. The hybrid peptide containing the mutated sequence (SFTI-Mut, GRCTRGRGLACFPD-NH2; Ki = 14 µM) presented an apparent inhibition constant (Ki(app)) for Pr2 proteases ?20-fold lower than the control peptide containing the original HiTI sequence (SFTI-HiTI, GRCTRKSDLSCFPD-NH2; Ki = 259 µM). In conclusion, the present work enabled the selection of a specific HiTI mutant for Pr2 proteases of M. anisopliae fungus using a HiTI combinatorial library on M13 phage surface. Selection of strong binders by phage display and their validation as inhibitors using synthetic hybrid peptides proved to be a powerful technique to generate specific serine protease inhibitors suitable for studies of drug design and enzyme-inhibitor interaction.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1875-5402
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
13
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
829-35
pubmed:dateRevised
2011-9-6
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Validation of phage display method for protease inhibitor selection using synthetic hybrid peptides.
pubmed:affiliation
Department of Biochemistry, Federal University of São Paulo (UNIFESP), Rua Três de Maio 100, 04044-020 São Paulo, SP, Brazil.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Validation Studies