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

Author:

BORASTON Alisdair B.123,WARREN R. Antony J.12,KILBURN Douglas G.123

Affiliation:

1. The Protein Engineering Network of Centres of Excellence, PENCE Inc., National Business Centre, 750 Heritage Medical Research Centre, Edmonton, Alberta, Canada T6G 2S2

2. Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

3. The Biotechnology Laboratory, 237-6174 University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

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 GlcNAc2Man8 to GlcNAc2Man14. 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 (Ka) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The Ka 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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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