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|Title:||Engineering molecular recognition of endoxylanase enzymes and their inhibitors through phage display||Authors:||Beliën, Tim
Van Campenhout, Steven
Bosch, An Vanden
Bourgois, Tine M.
Courtin, Christophe M.
Delcour, Jan A.
|Issue Date:||2007||Publisher:||JOHN WILEY & SONS LTD||Source:||JOURNAL OF MOLECULAR RECOGNITION, 20(2). p. 103-112||Abstract:||Specific binding of interacting proteins generally depends on a limited set of amino acid residues located at the contact interface. We have applied a phage-display-based screening method to simultaneously evaluate the role of multiple residues of endo-beta-1,4-xylanase enzymes in conferring binding specificity towards two different endoxylanase inhibitors. Seven residues of the two beta-strand 'thumb' region of Trichoderma longibrachiatum endo-beta-1,4-xylanase XynII were targeted for randomization. The generated combinatorial library representing 62 208 site-directed variants was displayed on the surface of filamentous phage and selected against xylanase inhibitor protein (XIP) and Triticum aestivum xylanase inhibitor (TAXI). DNA sequence analysis of phagemid panning isolates provided information on the occurrence of particular amino acids at distinct positions. In particular, residues at positions 124 (Asn) and 131 (Thr) were found to be critical for specific inhibitor binding. These residue predictions derived from the combinatorial exploration of the thumb region and accompanying sequence analyses were experimentally confirmed by testing the inhibitor sensitivity of a limited set of recombinantly expressed XynII mutants. In addition, we successfully altered the inhibition susceptibility of the bacterial Bacillus subtilis endoxylanase XynA from XIP-insensitive to XIP-sensitive. Copyright (c) 2007 John Wiley & Sons, Ltd.||Notes:||Katholieke Univ Leuven, Lab Food Chem & Biochem, B-3001 Louvain, Belgium. Katholieke Univ Leuven, Lab Gene Technol, B-3001 Louvain, Belgium. Univ Limburg, Biomed Res Inst, B-3590 Diepenbeek, Belgium.Belien, T, Katholieke Univ Leuven, Lab Food Chem & Biochem, Kasteelpk Arenberg 21, B-3001 Louvain, Belgium.firstname.lastname@example.org||Keywords:||combinatorial library; phage display; endoxylanase; inhibition specificity||Document URI:||http://hdl.handle.net/1942/4078||Link to publication:||http://doi.wiley.com/10.1002/jmr.818||ISSN:||0952-3499||e-ISSN:||1099-1352||ISI #:||000246169000005||Category:||A1||Type:||Journal Contribution||Validations:||ecoom 2008|
|Appears in Collections:||Research publications|
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