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PEDS Advance Access originally published online on September 9, 2005
Protein Engineering Design and Selection 2005 18(10):497-501; doi:10.1093/protein/gzi055
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

N-Glycosidase–carbohydrate-binding module fusion proteins as immobilized enzymes for protein deglycosylation

Emily M. Kwan1,2, Alisdair B. Boraston1,2,3,4,5, Bradley W. McLean1,2,3,4,6, Douglas G. Kilburn1,2,3 and R. Antony J. Warren1,2,7

1The Protein Engineering Network of Centres of Excellence, 750 Heritage Medical Research Centre, Edmonton, AB, T6G 2S2 and 2Department of Microbiology and Immunology and 3The Biotechnology Laboratory, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada 4A.B.Boraston and B.W.McLean contributed equally to this work 5Present address: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8P 5C2, Canada 6Present address: Twinstrand Therapeutics, 8081 Lougheed Highway, Burnaby, BC, V5A 1W9, Canada

7 To whom correspondence should be addressed. E-mail: rajw{at}interchange.ubc.ca

A carbohydrate-binding module (CBM) was fused to the N-termini of mannosyl-glycoprotein endo-ß-N-acetylglucosaminidase (EndoF1) and peptide N-glycosidase F (PNGaseF), two glycosidases from Chryseobacterium meningosepticum that are used to remove N-linked glycans from glycoproteins. The fusion proteins CBM–EndoF1 and CBM–PNGaseF also carry a hexahistidine tag for purification by immobilized metal affinity chromatography after production by Escherichia coli. CBM–EndoF1 is as effective as native EndoF1 at deglycosylating RNaseB; the glycans released by both enzymes are identical. Like native PNGaseF, CBM–PNGaseF is active on denatured but not on native RNaseB. Both fusion proteins are as active on RNaseB when immobilized on cellulose as they are in solution. They retain activity in the immobilized state for at least 1 month at 4°C. The hexahistidine tag can be removed with thrombin, leaving the CBM as the only affinity tag. The CBM can be removed with factor Xa if required.

Keywords: carbohydrate-binding module/fusion proteins/N-glycosidase

Received June 21, 2005; revised July 21, 2005; accepted August 3, 2005.

Edited by Pang-Chui Shaw


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