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. 2001 Sep 1;358(Pt 2):423–430. doi: 10.1042/0264-6021:3580423

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis.

A B Boraston 1, R A Warren 1, D G Kilburn 1
PMCID: PMC1222075  PMID: 11513741

Abstract

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc(2)Man(8) to GlcNAc(2)Man(14). The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (K(a)) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The K(a) of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

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Selected References

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