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Expression in Escherichia coli of the Cellulomonas fimi Structural Gene for Endoglucanase B

Expression in Escherichia coli of the Cellulomonas fimi Structural Gene for Endoglucanase B

Applied and Environmental Microbiology 54(2): 518-523

ISSN/ISBN: 0099-2240

PMID: 16347564

Endoglucanase B (EB) of Cellulomonas fimi has an M(r) of 110,000 when it is produced in Escherichia coli. The level of expression of the cenB gene (encoding EB) was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. EB was purified to homogeneity from the periplasmic fraction of E. coli in one step by affinity chromatography on microcrystalline cellulose (Avicel). Alignment of the NH(2)-terminal amino acid sequence with the partial nucleotide sequence of a fragment of C. fimi DNA showed that EB is preceded by a putative signal polypeptide of 33 amino acids. The signal peptide functions and is processed correctly in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of beta-galactosidase. The intact EB polypeptide is not required for enzymatic activity. Active polypeptides with M(r)s of 95,000 and 82,000 also appear in E. coli, and a deletion mutant of cenB encodes an active polypeptide with an M(r) of 72,000.

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Accession: 005429842

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