Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris

A stable GS115 Pichia pastoris recombinant strain was constructed tosecrete a truncated form of the human alpha(1,3/4) fucosyltransferase(amino acids 45-361). Enzyme production resulted from a secretory pathwaybased on the pre-pro- alpha mating factor signal sequence of the yeastSaccharomyces cerevisiae . Following its transit through the Golgiapparatus, the enzyme accumulated in the periplasmic space before itsrelease in the culture broth (about 30 mg/l). Cell-enclosed enzyme (approximately 0.16%) proved to be fairly stable for many freezing andthawing cycles and could be used several times as an immobilized catalyst.Soluble enzyme (>99.8%) representing the main protein of the culturebroth (10%) has been characterized by Western-blotting, substratespecificities and kinetic parameters. The two forms (cell- enclosed andsoluble) of recombinant enzyme may be used for in vitro synthesis ofLewisadeterminants.