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 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.
Gallet
P F
PF
Institut de Biotechnologie, Université de Limoges, France, INSERM U-178, Université de Paris-Sud XI, 94807 Villejuif, France.
Vaujour
H
H
Petit
J M
JM
Maftah
A
A
Oulmouden
A
A
Oriol
R
R
Le Narvor
C
C
Guilloton
M
M
Julien
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D005647:Fucosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013379:Substrate Specificity
D010446:Peptide Fragments; D011994:Recombinant Proteins; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase