Skip to main content

Downstream processing of a plant-made recombinant human therapeutic enzyme

Resource type
Thesis type
(Thesis) M.Sc.
Date created
2014-07-25
Authors/Contributors
Abstract
Mucopolysaccharidosis I is a lysosomal storage disease that is characterized by a deficiency of the lysosomal hydrolase alpha-L-iduronidase. Enzyme replacement therapy with purified, recombinant IDUA is one treatment option available for individuals that inherit this disease. The goal of my thesis was to engineer a transgenic line of Arabidopsis thaliana that could express, in the seed tissue, human iduronidase with a therapeutically acceptable N-glycan profile. Human iduronidase was expressed at 5.7% of total soluble protein (TSP) in the Arabidopsis cgl mutant line, which is deficient in N-acetylglucosamine transferase I, and at 1.5% of TSP in the Arabidopsis Golgi-Mannosidase I (GM-I) knockout line. The iduronidase purified from both lines was able to hydrolyze the fluorescent iduronide compound, 4-methylumbelliferyliduronide (4-MUI), at a rate comparable to that of iduronidase produced in Chinese Hamster Ovary cells, commercially available as Aldurazyme. Both plant-derived forms of iduronidase possessed primarily high-mannose N-glycans (at 95% for cgl-iduronidase and 100% for GM1-iduronidase); however, the dominant glycoform in the N-glycan profile of cgl-iduronidase was Man5GlcNAc2, whereas most of the N-glycans on GM1-iduronidase contained six to eight mannose sugars. Additionally, cgl-iduronidase contained a small percentage of complex glycans (and thus potentially immunogenic xylose and/or fucose sugars), which were not found on GM1-iduronidase. Surprisingly GM1-iduronidase did not contain an N-glycan at Asn336, which is normally present on the native human iduronidase and on the recombinant iduronidase of cgl Arabidopsis seeds and Chinese hamster ovary cells. Of importance to creating a product that is of therapeutic value, another goal was to determine whether the plant-produced iduronidase was amenable to in vitro phosphorylation to create the mannose-6-phosphate tag necessary for sequestration and lysosomal delivery in human cells. Both plant-derived forms of the enzymes were able to be phosphorylated in vitro by the UDP-GlcNAc:lysosomal enzyme (GlcNAc)-1-phosphotransferase at a rate similar to that of Aldurazyme.
Document
Identifier
etd8621
Copyright statement
Copyright is held by the author.
Permissions
The author granted permission for the file to be printed and for the text to be copied and pasted.
Scholarly level
Supervisor or Senior Supervisor
Thesis advisor: Kermode, Allison
Member of collection
Download file Size
etd8621_OPierce.pdf 2.74 MB

Views & downloads - as of June 2023

Views: 18
Downloads: 1