Development of recombinant, immobilised β-1,4-mannosyltransferase for use as an efficient tool in the chemoenzymatic synthesis of N-linked oligosaccharides

Abstract

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 Manβ4GlcNAcβ4GlcNAc (Gn2M) core trisaccharide has proved to be a major obstacle because of the inherent difficulties associated with the synthesis of β-mannosides. Here we report the overproduction in Escherichia coli of full-length and transmembrane-deleted yeast β-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 K m of 14 μM for the synthetic acceptor substrate analogue, phytanyl-pyrophosphoryl-α- N, N′-diacetylchitobioside (PPGn2), under saturating donor conditions. This figure is comparable to those previously reported for native and recombinant yeast β-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.