Properties conferred on Clostridium thermocellum endoglucanase CelC by grafting the duplicated segment of endoglucanase CelD.
The DNA sequence encoding the duplicated 22 amino acid segment of Clostridium thermocellum endoglucanase CelD was fused to the 3'-terminus of the celC gene encoding C.thermocellum endoglucanase CelC. The presence of the duplicated segment endowed CelC with the capacity to form cytoplasmic inclusion bodies containing active enzyme when the hybrid gene was expressed in Escherichia coli. Inclusion body formation prevented proteolytic cleavage of the duplicated segment. The intact hybrid protein CelC-Cel'D was purified from inclusion bodies and characterized. In contrast to CelC, CelC-Cel'D was able to bind to CipA, a protein acting as a scaffolding component of the C.thermocellum cellulase complex (cellulosome). However, the catalytic properties of CelC-Cel'D were similar to those of CelC. These results suggest that foreign proteins tagged with the duplicated segment could be incorporated into the cellulosome in order to modify the enzymatic properties of the complex. The formation of inclusion bodies by proteins carrying the duplicated segment may also prove a convenient means of purifying cloned gene products that are sensitive to proteolytic degradation.
Tokatlidis
K
K
Unité de Physiologie Cellulaire and URA 1300 CNRS, Institut Pasteur, Paris, France.
Dhurjati
P
P
Béguin
P
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002118:Calcium / Q000378:metabolism; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D011232:Chemical Precipitation; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D002479:Inclusion Bodies / Q000201:enzymology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002482:Cellulose; C083712:celC protein, bacteria; C087879:endoglucanase CelD; D001617:beta-Glucosidase; D002480:Cellulase; D002118:Calcium