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 oligos accharides found in eukaryotic glycoproteins is an important step towards t he synthesis of homogeneous neoglycoproteins, So far, however, the convenie nt generation of the Man beta 4GlcNAc beta 4GlcNAc (Gn2M) core trisaccharid e has proved to be a major obstacle because of the inherent difficulties as sociated with the synthesis of beta-mannosides. Here we report the overprod uction in Escherichia coli of full-length and transmembrane-deleted yeast b eta-1,4-mannosyltransferases as novel N-terminal fusions bearing a decahist idinyl sequence and the minimal human Myc epitope. The recombinant enzymes were highly active and were amenable to immobilisation by nickel(II) chelat ion and to immunodetection with an anti-Myc monoclonal antibody. The immobi lised, transmembrane-deleted enzyme exhibited an apparent K-m of 14 mu M fo r the synthetic acceptor substrate analogue, phytanyl-pyrophosphoryl-alpha- N,N'-diacetylchitobioside (PPGn2), under saturating donor conditions. This figure is comparable to these previously reported for native and recombinan t yeast beta-1,4-mannosyltransferases with, respectively, the natural dolic hyl-linked acceptor and PPGn2.. The validity of the reaction product was co nfirmed by chromatographic and spectroscopic analysis. (C) 1999 Elsevier Sc ience B.V. All rights reserved.