Glycoside hydrolase carbohydrate-binding modules as molecular probes for the analysis of plant cell wall polymers.
Novel molecular probes have been developed for the analysis and detection of polysaccharides in plant cell walls using carbohydrate-binding modules (CBMs) derived from modular glycoside hydrolases belonging to families 2a, 6, and 29. Recombinant forms of these proteins containing his-tags, in conjunction with anti-his-tag detection, provide a flexible system that utilizes CBMs as molecular probes in a range of applications. Assays for the rapid analysis of the binding of CBMs to polysaccharides and oligosaccharides using nitrocellulose-based CBM macroarrays and microtiter plate-based CBM capture and competitive-inhibition assays are described. We also demonstrate the use of CBMs with his-tags for the localization of their target ligands in planta. The generation of molecular probes from other families of CBMs will dramatically increase the repertoire of molecular probes available to determine the developmental and functional aspects of plant cell walls.
McCartney
Lesley
L
Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK.
Gilbert
Harry J
HJ
Bolam
David N
DN
Boraston
Alisdair B
AB
Knox
J Paul
JP
D001704:Biopolymers / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry; D002473:Cell Wall / Q000737:chemistry*; D003101:Collodion; D006026:Glycoside Hydrolases / Q000378:metabolism*; D008024:Ligands; D015336:Molecular Probe Techniques; D059828:Plant Cells; D010944:Plants / Q000737:chemistry*
D001704:Biopolymers; D002241:Carbohydrates; D008024:Ligands; D003101:Collodion; D006026:Glycoside Hydrolases