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The cellulose‐binding domain (CBD Cex ) of an exoglucanase from Cellulomonas fimi : Production in Escherichia coli and characterization of the polypeptide
Author(s) -
Ong Edgar,
Gilkes Neil R.,
Miller Robert C.,
Warren R. Antony J.,
Kilburn Douglas G.
Publication year - 1993
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260420402
Subject(s) - escherichia coli , chemistry , biochemistry , cellulose , amino acid , cellulase , stereochemistry , gene
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 Nhe l site introduced during plasmid construction. CBD Cex bound to a variety of β‐1, 4‐glycans with different affinities and saturation levels. Adsorption to bacterial microcrystalline cellulose was dependent on the temperature, but not on the pH. © 1993 John Wiley & Sons, Inc.