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Engineering molecular recognition of endoxylanase enzymes and their inhibitors through phage display
Author(s) -
Beliën Tim,
Van Campenhout Steven,
Bosch An Vanden,
Bourgois Tine M.,
Rombouts Sigrid,
Robben Johan,
Courtin Christophe M.,
Delcour Jan A.,
Volckaert Guido
Publication year - 2007
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.818
Subject(s) - biochemistry , phage display , bacillus subtilis , xylanase , biology , phagemid , panning (audio) , enzyme , protein engineering , amino acid , chemistry , bacteriophage , escherichia coli , genetics , bacteria , peptide , gene , paleontology , zoom , lens (geology)
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‐β‐1,4‐xylanase enzymes in conferring binding specificity towards two different endoxylanase inhibitors. Seven residues of the two β‐strand ‘thumb’ region of Trichoderma longibrachiatum endo‐β‐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 © 2007 John Wiley & Sons, Ltd.