Functional display of family 11 endoxylanases on the surface of phage M13.
Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.
Beliën
T
T
Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium.
Hertveldt
K
K
Van den Brande
K
K
Robben
J
J
Van Campenhout
S
S
Volckaert
G
G
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D017104:Bacteriophage M13 / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
D008565:Membrane Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases