Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector.
Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. The enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. To perform more extensive biochemical characterization and to develop new approaches for enzyme therapy, a method of producing and purifying recombinant alpha-galactosidase A suitable for scale-up manufacture for use in humans is needed. Previously, a catalytically active recombinant human alpha-galactosidase A was expressed using a baculovirus vector and purified using conventional chromatography. However, the level of expression was too low to permit economical production and the chromatographic techniques used for enzyme purification were not suitable for enzyme to be used in humans. Therefore, the cDNA of the enzyme was cloned to an improved baculovirus vector and the enzyme was expressed in a 15-liter bioreactor using optimized growth conditions. Infection of insect cells by the baculovirus resulted in a significant fivefold increase in the level of secreted recombinant alpha-galactosidase A activity that is compatible with economic manufacturing. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (Poros 20-CM, Poros 20-HQ) and hydrophobic chromatography (Toso-ether, Toso-butyl) using the BioCAD HPLC workstation. These chromatographic steps are readily scalable to larger volumes and are appropriate for the purification of the recombinant human alpha-galactosidase A to be used in clinical trials of enzyme replacement therapy for Fabry disease patients.
Chen
Y
Y
Department of Chemistry, City College of New York, 138th Street and Convent Avenue, New York, New York 10031, USA.
Jin
M
M
Goodrich
L
L
Smith
G
G
Coppola
G
G
Calhoun
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D019149:Bioreactors; D002241:Carbohydrates / Q000032:analysis; D002845:Chromatography; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D000795:Fabry Disease / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D005347:Fibroblasts; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000821:virology; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D002241:Carbohydrates; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D000519:alpha-Galactosidase