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

1998; Oxford University Press; Volume: 8; Issue: 9 Linguagem: Inglês

10.1093/glycob/8.9.919

1460-2423

Paul‐François Gallet, H. Vaujour, Jean‐Michel Petit, Abderrahman Maftah, Ahmad Oulmouden, Rafaël Oriol, Christine Le Narvor, Michel Guilloton, R. Julien,

Glycosylation and Glycoproteins Research

A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human α(1,3/4) fucosyltransferase (amino acids 45–361). Enzyme production resulted from a secretory pathway based on the pre-pro- α 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 (∼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 Lewisa determinants.