Expression and characterization of recombinant human alpha-galactosidase A.
Item
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Title
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Expression and characterization of recombinant human alpha-galactosidase A.
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Identifier
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AAI9108121
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identifier
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9108121
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Creator
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Ioannou, Yiannis Andreas.
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Contributor
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Advisers: David F. Bishop | Robert J. Desnick
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Date
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1990
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Genetics | Biology, Molecular
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Abstract
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Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, {dollar}\alpha{dollar}-galactosidase A ({dollar}\alpha{dollar}-Gal A). In order to characterize the normal enzyme and to evaluate the clinical effectiveness of enzyme replacement therapy, efforts were directed to produce large quantities of human recombinant {dollar}\alpha{dollar}-Gal A. A full-length {dollar}\alpha{dollar}-Gal A cDNA was inserted into the mammalian expression vector p91023(B) in front of the amplifiable dihydrofolate reductase (DHFR) cDNA. This construct was introduced into DG44 {dollar}dhfr\sp{lcub}-{rcub}{dollar} CHO cells. Selected subclones were grown in increasing concentrations of methotrexate (MTX, 0.02 to 1.3 {dollar}\mu{dollar}M) resulting in co-amplification of DHFR and {dollar}\alpha{dollar}-Gal A cDNAs. At a MTX concentration of 1.3 {dollar}\mu{dollar}M, 10{dollar}\sp7{dollar} cells secreted {dollar}\sim{dollar}15,000 U/ml culture media/day. Using a hollow fiber bioreactor, up to 10 mg of enzyme protein was secreted per day.;The secreted {dollar}\alpha{dollar}-Gal A was purified by affinity chromatography for characterization of various physical and kinetic properties. The recombinant enzyme had a pI of 3.9, a pH optimum of 4.6, a km of 1.9 mM toward 4-methylumbelliferyl-{dollar}\alpha{dollar}-D-galactopyranoside and rapidly hydrolyzed globotriaosylceramide, the natural glycosphingolipid substrate. Pulse-chase studies indicated that the recombinant enzyme assumed its secondary structure in {dollar}<{dollar}3 min, was in the Golgi by 5 min where it became Endo H resistant, and was secreted into the media by 45-60 min. Labeling studies revealed that both the intracellular and secreted forms were phosphorylated. Further analysis revealed the presence of three {dollar}N{dollar}-linked oligosaccharide chains, two high-mannose type (Endo H sensitive) and one complex type. Analyses of the Endo H released oligosaccharides revealed that one had two phosphate residues and it specifically bound to immobililzed mannose-6-phosphate receptors while the other was a hybrid structure containing sialic acid. The secreted form of {dollar}\alpha{dollar}-Gal A was taken up by cultured Fabry fibroblasts by a saturable process that was blocked in the presence of 2 mM mannose-6-phosphate. The availability of large amounts of soluble, active recombinant {dollar}\alpha{dollar}-Gal A which is similar in structure to the native enzyme isolated from plasma will permit further comparison to the native enzyme forms and the clinical evaluation of enzyme replacement in Fabry disease.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
