The aim of the project is to
develop novel analytical tools based on carbohydrate binding modules (CBMs) for
investigate the carbohydrate composition and spatial distribution on pulp fibre
involved in carbohydrate metabolism and turnover often have modular structures
with a catalytic domain linked to one or more substrate-binding modules. The
first CBMs isolated showed affinity towards cellulose. Later on other CBMs have
been found, with affinity towards other cell wall carbohydrates such as xylan,
chitin and mannan. Since these are recognition elements evolved in nature,
their specificity is better than for example that of antibodies generated
against carbohydrates, which are often poorly antigenic.
Project description: Different CBMs are expressed in E. coli, fused to the engineered double ZZ-domain of the staphylococcal protein A. The ZZ-domain provides a specific binding site of antibodies and thus allows direct immunogold labeling of the fusion proteins (see Fig 1). Since no secondary antibodies are needed, better resolution can be achieved when transmisson electron microscopy (TEMI is used for imaging). When CBMs with different specificities are fused to the ZZ-module, the different carbohydrates located on pulp fibre surfaces can be mapped with very good precision. The project involves cloning and expression of CBMs with known substrate specificity as well as the characterization of CBMs with so far unknown binding specificity.