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Glycobiology, Vol 8, 919-925, Copyright © 1998 by Society for Glycobiology

ORIGINAL ARTICLES

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

PF Gallet, H Vaujour, JM Petit, A Maftah, A Oulmouden, R Oriol, C Le Narvor, M Guilloton and R Julien
Institut de Biotechnologie, Universite de Limoges, France, INSERM U- 178, Universite de Paris-Sud XI, 94807 Villejuif, France.

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