Expression, purification and crystallization of a cohesin domain from the cellulosome of Clostridium thermocellum.
The cellulosome of the cellulolytic bacterium, Clostridium thermocellum, is a multi-enzyme complex in which the enzymatic (cellulolytic) subunits are attached to a unique nonhydrolytic subunit called scaffoldin. The attachment is mediated by two mutually interacting domains: namely multiple cohesin domains on the scaffoldin subunit and a dockerin domain on each of the enzymatic subunits. Knowledge of the three-dimensional structure of each of the interacting components would be critical to a better understanding of the cohesin-dockerin interaction at the molecular level. In this report, we describe the purification of one of the nine cohesin domains of the scaffoldin subunit from C. thermocellum. A DNA segment containing the cohesin 2 sequence was fused to a hexa-histidine tag, and the resultant construct was expressed in Escherichia coli. The expressed peptide was efficiently isolated by metal-chelate affinity chromatography. The purified recombinant form of the cohesin was crystallized pending determination of its structure.
Yaron
S
S
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa, Israel.
Shimon
L J
LJ
Frolow
F
F
Lamed
R
R
Morag
E
E
Shoham
Y
Y
Bayer
E A
EA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D003460:Crystallization; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D002480:Cellulase