Endoglucanase E, produced at high level in Escherichia coli as a lacZ′ fusion protein, is part of the Clostridium thermocellum cellulosome

• Published in: Enzyme and microbial technology Vol. 12; no. 9; pp. 656 - 662
• Main Authors: Hazlewood, Geoffrey P., Davidson, Keith, Clarke, Jonathan H., Durrant, Alastair J., Hall, Judith, Gilbert, Harry J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.1990
• Subjects: Amino Acid Sequence; Base Sequence; Cellulase - biosynthesis; Cellulase - genetics; Cellulase - secretion; cloning; Clostridium - enzymology; Clostridium - genetics; Clostridium thermocellum; DNA Mutational Analysis; Endoglucanase; endoglucanase E; Escherichia coli; Escherichia coli - metabolism; gene fusion; genes; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Protein Processing, Post-Translational; Protein Sorting Signals - physiology; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - secretion; Structure-Activity Relationship; Subcellular Fractions - enzymology; Endoglucanase; Clostridium thermocellum; Escherichia coli
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/0141-0229(90)90004-A

The Clostridium thermocellum celE gene was expressed at high level in Escherichia coli TG1 when placed under the transcriptional and translational control of lacZ′ in pUC18; in the presence of a multicopy plasmid (pNM52) containing the lacI q gene, expression of full-length and truncated forms of celE was regulated by isopropyl-β- d-thiogalactopyranoside. Catalytically active endoglucanase E (EGE) produced by E. coli was subject to proteolytic processing. The main protein species produced from full-length and truncated forms of celE was around 40 kDa in size and had an N-terminal amino acid sequence corresponding to that derived for mature EGE from the nucleotide sequence; in addition, larger species of about 75 kDa, presumably corresponding to full-size EGE, were produced by E.coli containing the full-length celE gene. Even after removal of the signal peptide sequence, EGE produced by E. coli was secreted into the periplasm. Up to 157 bp could be deleted from the 5′ end of the celE gene without affecting the catalytic activity of EGE produced by E. coli. A polypeptide of M , 86 kDa, immunoreactive with anti-EGE antiserum, was demonstrated in the high-molecular-weight, cellulose-binding multiprotein aggregate recoverable from C. thermocellum culture supernatant.