Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum.
The major cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum YS was cloned and overexpressed in Escherichia coli. The expressed protein was purified efficiently by a modification of a novel procedure termed affinity digestion. The properties of the purified polypeptide were compared with those of a related CBD derived from a cellulosome-like complex of a similar (but mesophilic) clostridial species, Clostridium cellulovorans. The binding properties of the two proteins with their common substrate were found to be very similar. Despite the similarity in the amino acid sequences of the two CBDs, polyclonal antibodies raised against the CBD from C. thermocellum failed to interact with the protein from C. cellulovorans. Chemical modification of the single cysteine of the CBD had little effect on the binding to cellulose. Biotinylation of this cysteine allowed the efficient binding of avidin to cellulose, and the resultant matrix is appropriate for use as a universal affinity system.
Morag
E
E
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Lapidot
A
A
Govorko
D
D
Lamed
R
R
Wilchek
M
M
Bayer
E A
EA
Shoham
Y
Y
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001360:Avidin; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000737:chemistry* / Q000145:classification / Q000378:metabolism / Q000648:ultrastructure; D004790:Enzyme Induction; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry; D015388:Organelles / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000345:Affinity Labels; D001426:Bacterial Proteins; D002352:Carrier Proteins; C080369:CipA protein, Clostridium; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D001360:Avidin; C074074:cellulose binding protein A, Clostridium cellulovorans; D001710:Biotin; D002482:Cellulose