Expression of eukaryotic glycosyltransferases in the yeast Pichia pastoris

Abstract

The methylotrophic yeast Pichia pastoris is often used as an organism for the heterologous expression of proteins and has been used already for production of a number of glycosyltransferases involved in the biosynthesis of N- and O-linked oligosaccharides. In our recent studies, we have examined the expression in P. pastoris of Arabidopsis thaliana and Drosophila melanogaster core α1,3-fucosyltransferases (EC 2.4.1.214), A. thaliana β1,2-xylosyltransferase (EC 2.4.2.38), bovine β1,4-galactosyltransferase I (EC 2.4.1.38), D. melanogaster peptide O-xylosyltransferase (EC 2.4.2.26), D. melanogaster and Caenorhabditis elegans β1,4-galactosyltransferase VII (SQV-3; EC 2.4.1.133) and tomato Lewis-type α1,4-fucosyltransferase (EC 2.4.1.65). Temperature, cell density and medium formulation have varying effects on the amount of activity resulting from expression under the control of either the constitutive glyceraldehyde-3-phosphate dehydrogenase ( GAP) or inducible alcohol oxidase ( AOX1) promoters. In the case of the A. thaliana xylosyltransferase these effects were most pronounced, since constitutive expression at 16 °C resulted in 30-times more activity than inducible expression at 30 °C. Also, the exact nature of the constructs had an effect; whereas soluble forms of the A. thaliana xylosyltransferase and fucosyltransferase were active with N-terminal pentahistidine tags (in the former case facilitating purification of the recombinant protein to homogeneity), a C-terminally tagged form of the A. thaliana fucosyltransferase was inactive. In the case of D. melanogaster β1,4-galactosyltransferase VII, expression with a yeast secretion signal yielded no detectable activity; however, when a full-length form of the enzyme was introduced into P. pastoris, an active secreted form of the protein was produced.