Development of recombinant, immobilised beta-1,4-mannosyltransferase for use as an efficient tool in the chemoenzymatic synthesis of N-linked oligosaccharides.
The preparation of the conserved core structure of asparagine-linked oligosaccharides found in eukaryotic glycoproteins is an important step towards the synthesis of homogeneous neoglycoproteins. So far, however, the convenient generation of the Manbeta4GlcNAcbeta4GlcNAc (Gn2M) core trisaccharide has proved to be a major obstacle because of the inherent difficulties associated with the synthesis of beta-mannosides. Here we report the overproduction in Escherichia coli of full-length and transmembrane-deleted yeast beta-1, 4-mannosyltransferases as novel N-terminal fusions bearing a decahistidinyl sequence and the minimal human Myc epitope. The recombinant enzymes were highly active and were amenable to immobilisation by nickel(II) chelation and to immunodetection with an anti-Myc monoclonal antibody. The immobilised, transmembrane-deleted enzyme exhibited an apparent Km of 14 microM for the synthetic acceptor substrate analogue, phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside (PPGn2), under saturating donor conditions. This figure is comparable to those previously reported for native and recombinant yeast beta-1, 4-mannosyltransferases with, respectively, the natural dolichyl-linked acceptor and PPGn2. The validity of the reaction product was confirmed by chromatographic and spectroscopic analysis.
Revers
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The Edinburgh Centre for Protein Technology, Department of Chemistry, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JJ, UK.
Bill
R M
RM
Wilson
I B
IB
Watt
G M
GM
Flitsch
S L
SL
D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D004800:Enzymes, Immobilized / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008364:Mannosyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D004800:Enzymes, Immobilized; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase