title: Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris.
abstract: Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. This enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. An improved method of production of recombinant alpha-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human alpha-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human alpha-galactosidase A. Recombinant human alpha-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25 degrees C, but not at the standard growth temperature of 30 degrees C. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant alpha-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates.
authors.affiliation: Department of Chemistry, City College of New York, 138th Street and Convent Avenue, New York, New York 10031, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: M
authors.initials: M
authors.lastname: Jin
authors.forename: T
authors.initials: T
authors.lastname: Egborge
authors.forename: G
authors.initials: G
authors.lastname: Coppola
authors.forename: J
authors.initials: J
authors.lastname: Andre
authors.forename: D H
authors.initials: DH
authors.lastname: Calhoun
mesh_terms: D019149:Bioreactors; D002241:Carbohydrates / Q000032:analysis; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D000795:Fabry Disease / Q000378:metabolism; D005285:Fermentation; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D011994:Recombinant Proteins; D000519:alpha-Galactosidase