Overproduction, purification and characterization of the cellulose-binding domain of the Erwinia chrysanthemi secreted endoglucanase EGZ.
EGZ is the major endoglucanase secreted by Erwinia chrysanthemi. Functional characterization indicates that it is made of a catalytic N-terminal domain linked to a C-terminal cellulose-binding domain (CBD) by a Ser/Thr-rich linker. A chimeric plasmid, in which the CBD-encoding region was fused downstream of the ompA signal sequence, was constructed and introduced into Escherichia coli. This allowed for the production of processed and disulfide-bonded CBD, mostly recovered from the culture supernatant of E. coli. One-dimensional NMR analysis of the purified CBD reveals that it folds into a well-structured domain. Moreover, comparison with the one-dimensional NMR analysis of full-length EGZ strongly suggests that the CBD folds autonomously, providing experimental support for the existence of domains of EGZ.
Brun
E
E
LCB-CNRS, Marseille, France.
Gans
P
P
Marion
D
D
Barras
F
F
D001665:Binding Sites; D002480:Cellulase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D016974:Dickeya chrysanthemi / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D002482:Cellulose; C080692:endoglucanase Z; D002480:Cellulase