Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum

Abstract

The major cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum YS was cloned and overexpressed in Escherichia coli. The expressed protein was purified efficiently by a modification of a novel procedure termed affinity digestion. The properties of the purified polypeptide were compared with those of a related CBD derived from a cellulosome-like complex of a similar (but mesophilic) clostridial species, Clostridium cellulovorans. The binding properties of the two proteins with their common substrate were found to be very similar. Despite the similarity in the amino acid sequences of the two CBDs, polyclonal antibodies raised against the CBD from C. thermocellum failed to interact with the protein from C. cellulovorans. Chemical modification of the single cysteine of the CBD had little effect on the binding to cellulose. Biotinylation of this cysteine allowed the efficient binding of avidin to cellulose, and the resultant matrix is appropriate for use as a universal affinity system.

Cellulosomal carbohydrate‐binding module from Clostridium josui binds to crystalline and non‐crystalline cellulose, and soluble polysaccharides

“…4). At the first binding site, the association constant was 4.1 Â 10 6 M À1 , a value comparable to those of other scaffoldin CBMs [12,21] (Table 1). An association constant could not be determined for the second binding site, indicating that the value was too small to resolve by this model.…”

Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes ofAcetivibrio cellulolyticusandClostridium thermocellum

“…DNA fragments encoding either CBMs or Cel9B were amplified by PCR from A. cellulolyticus ATCC 33288 or C. thermocellum YS genomic DNA, using appropriate primers as listed in Table 1. Plasmid pET9d-CBM3a, encoding the CBM3a domain of C. thermocellum CipA, was a modification of that described previously (Morag et al, 1995). The desired DNA was cloned in Escherichia coli XL1-Blue.…”

Cohesin‐dockerin microarray: Diverse specificities between two complementary families of interacting protein modules

“…integrated into a central multimodular scaffolding subunit (scaffoldin) [4,5], by virtue of a specialized type of cohesin module, present in multiple copies on the scaffoldin, which binds tightly to a complementary dockerin module borne by each of the cellulosomal components. The scaffoldin also harbors a cellulose-binding module (CBM), which targets the cellulosome to its substrate [6,7], and a divergent dockerin of its own, which binds to selected cohesins of alternative scaffoldin(s) that anchor the cellulosome complex to the bacterial cell surface [8][9][10][11][12]. Cellulosome architecture is determined by specificities of the different cohesin-dockerin interactions, and their number and arrangement in the different scaffoldins and other cellulosomal subunits.…”