A family 2a carbohydrate-binding module suitable as an affinity tag for proteins produced in Pichia pastoris

The family 2a carbohydrate-binding module (CBM), Ce15ACBM2a, from the C-ter minus of Ce15A from Cellulomonas fimi, and Xyn10ACBM2a, the family 2a CBM f rom the C-terminus of Xyn10A from C. fimi, were compared as fusion partners for proteins produced in the methylotrophic yeast Pichia pastoris. G;ene f usions of murine stem-cell factor (SCF) with both CBMs were expressed in P. pastoris. The secreted SCF-Xyn10ACBM2a polypeptides were highly glycosylat ed and bound poorly to cellulose. In contrast, fusion of SCF to Ce15ACBM2a, which lacks potential N-linked glycosylation sites, resulted in the produc tion of polypeptides which bound tightly to cellulose. Cloning and expressi on of these CBM2a in P. pastoris without a fusion partner confirmed that N- linked glycosylation at several sites was responsible for the poor cellulos e binding. The nonglycosylated CBMs produced in E. coli had very similar ce llulose-binding properties. (C) 2001 Academic Press.