Endoglucanase E, produced at high level in Escherichia coli as a lacZ' fusion protein, is part of the Clostridium thermocellum cellulosome.
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 lacIq gene, expression of full-length and truncated forms of celE was regulated by isopropyl-beta-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 Mr 86 kDa, immunoreactive with anti-EGE antiserum, was demonstrated in the high-molecular-weight, cellulose-binding multiprotein aggregate recoverable from C. thermocellum culture supernatant.
Department of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, UK.
G P
GP
Hazlewood
K
K
Davidson
J H
JH
Clarke
A J
AJ
Durrant
J
J
Hall
H J
HJ
Gilbert
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology
D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002480:Cellulase