The gene fragment encoding the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi was subcloned and expressed in Escherichia coli. Transcription from the lac promoter coupled with translation from a consensus prokaryotic ribosome binding site led to the production of large quantities of CBD(Cex) (up to 25% total soluble cell protein). The polypeptide leaked into the culture supernatant (up to 50 mg . L(-1)), facilitating one-step purification by affinity chromatography on cellulose. The 11-kDa polypeptide reacted with Cex antiserum. Absence of free thiols indicated that the two Cys residues of CBD(Cex) form a disulfide bridge. It had the same N-terminal amino acid sequence as CBD(Cex) prepared from Cex by proteolysis, plus two additional N-terminal amino acid residues (Ala and Ser) encoded by the Nhel site introduced during plasmid construction. CBD(Cex) bound to a variety of beta-1, 4-glycans with different affinities and saturation levels. Adsorption to bacterial microcrystalline cellulose was dependent on the temperature, but not on the pH.
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