A family 2a carbohydrate-binding module suitable as an affinity tag for proteins produced in Pichia pastoris.
The family 2a carbohydrate-binding module (CBM), Cel5ACBM2a, from the C-terminus of Cel5A from Cellulomonas fimi, and Xyn10ACBM2a, the family 2a CBM from the C-terminus of Xyn10A from C. fimi, were compared as fusion partners for proteins produced in the methylotrophic yeast Pichia pastoris. Gene fusions of murine stem-cell factor (SCF) with both CBMs were expressed in P. pastoris. The secreted SCF-Xyn10ACBM2a polypeptides were highly glycosylated and bound poorly to cellulose. In contrast, fusion of SCF to Cel5ACBM2a, which lacks potential N-linked glycosylation sites, resulted in the production of polypeptides which bound tightly to cellulose. Cloning and expression of these CBM2a in P. pastoris without a fusion partner confirmed that N-linked glycosylation at several sites was responsible for the poor cellulose binding. The nonglycosylated CBMs produced in E. coli had very similar cellulose-binding properties.
Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
A B
AB
Boraston
B W
BW
McLean
M M
MM
Guarna
E
E
Amandaron-Akow
D G
DG
Kilburn
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D002846:Chromatography, Affinity; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019089:Stem Cell Factor / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D002482:Cellulose; D014995:Xylosidases; C407024:endoglucanase Cel5A; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase