Research Article

O-Glycosylation of a Recombinant Carbohydrate-Binding Module Mutant Secreted by Pichia pastoris
Alisdair B. Boraston^a-c, Linda E. Sandercock^a,b, R. Antony J. Warren^a,b, Douglas G. Kilburn<sup>a-c

a</sup>The Protein Engineering Network of Centres of Excellence, PENCE Inc., Edmonton;
^bDepartment of Microbiology and Immunology, and
^cBiotechnology Laboratory, University of British Columbia, Vancouver, Canada

Address of Corresponding Author

J Mol Microbiol Biotechnol 2003;5:29-36 (DOI: 10.1159/000068721)

  Key Words

• Cellulose-binding domain
• Carbohydrate-binding module
• CBD_Cex
• CBM2a
• Glycosylation
• Post-translational modification

  Abstract

Carbohydrate-binding modules (CBMs; previously called cellulose-binding domains) make excellent fusion partners for the immobilization or purification of polypeptides. However, their use in eukaryotic hosts has been limited by glycosylation, which interferes with the ability of the CBM to bind to cellulose. We have engineered the C-terminal carbohydrate-binding module from Cellulomonas fimi xylanase 10A such that it lacks N-glycosylation sites. This variant, called CBM2aNgly-, was produced and secreted by the methylotrophic yeast Pichia pastoris and found to be O-glycosylated. The O-linked glycans were composed entirely of mannose in a ratio of 1 mol of mannose to 4 mol of protein. The overall distribution of mannose on the O-glycosylated CBM mutant ranged from 1 to 9 mannose residues with the oligosaccharide sizes ranging from Man₁ to Man₄. MALDI-TOF (all matrix-assisted-laser-desorption time of flight) mass spectrometry (MS) was used to map the O-glycosylation to three regions of the polypeptide, each region having a maximum of 4 mannose residues attached to each. Glycans chemically released from CBM2aNgly- and analyzed by fluorophore-assisted carbohydrate electrophoresis were found to contain -1,2-, -1,3-, and -1,6-linkages. Significantly, the O-glycosylation did not influence binding, making CBM2aNgly- a suitable fusion partner for polypeptides produced in P. pastoris and other eukaryotic hosts.

Copyright © 2003 S. Karger AG, Basel

  Author Contacts

Alisdair B. Boraston
Department of Chemistry, University of York
Heslington, York, YO10 5DD (UK)
Tel. +44 1904 434529, Fax +44 1904 410519
E-Mail boraston@ysbl.york.ac.uk

  Article Information

Number of Print Pages : 8
Number of Figures : 6, Number of Tables : 1, Number of References : 23