EXPRESSION, PURIFICATION AND SUBUNIT-BINDING PROPERTIES OF COHESIN-2 AND COHESIN-3 OF THE CLOSTRIDIUM-THERMOCELLUM CELLULOSOME

The enzymatic subunits of the cellulosome of Clostridium thermocellum are integrated into the complex by a major non-catalytic polypeptide, called scaffoldin. Its numerous functional domains include a single ce llulose-binding domain (CBD) and nine subunit-binding domains, or cohe sin domains, Two of the cohesin domains, together with the adjacent CB D, have been cloned and expressed in Escherichia coli, and the recombi nant constructs were purified by affinity chromatography on a cellulos ic matrix. Both cohesin domains, which differ by about 30% in their pr imary structure, showed a similar binding profile to the cellulosomal subunits, Calcium ions enhanced dramatically this binding. Under the c onditions of the assay, only one major catalytic subunit of the cellul osome failed to bind to either cohesin domain. The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly,