Title: Engineering molecular recognition of endoxylanase enzymes and their inhibitors through phage display
Authors: Beliën, Tim ×
Van Campenhout, Steven
Vanden Bosch, An
Bourgois, Tine M
Rombouts, Sigrid
Robben, Johan
Courtin, Christophe
Delcour, Jan
Volckaert, Guido #
Issue Date: Mar-2007
Publisher: Heydon & Son
Series Title: Journal of Molecular Recognition vol:20 issue:2 pages: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.
ISSN: 0952-3499
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Clinical and Experimental Endocrinology
Biochemistry, Molecular and Structural Biology Section
Centre for Food and Microbial Technology
Division of Gene Technology (-)
× corresponding author
# (joint) last author

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