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

Glycobiology. 1998 Sep;8(9):919-25. doi: 10.1093/glycob/8.9.919.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Carbohydrate Sequence
  • Fucosyltransferases / biosynthesis*
  • Fucosyltransferases / genetics
  • Genetic Vectors
  • Humans
  • Molecular Sequence Data
  • Peptide Fragments / biosynthesis*
  • Peptide Fragments / genetics
  • Pichia / genetics
  • Protein Engineering
  • Recombinant Proteins / biosynthesis*
  • Substrate Specificity

Substances

  • Peptide Fragments
  • Recombinant Proteins
  • Fucosyltransferases
  • 3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase