1. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase.
- Author
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Branco Novo, Juliana, Morganti, Ligia, Moro, Ana Maria, Paes Leme, Adriana Franco, de Toledo Serrano, Solange Maria, Raw, Isaias, and Lee Ho, Paulo
- Subjects
GENETIC techniques ,GAUCHER'S disease treatment ,GENETIC engineering ,ANIMAL experimentation ,CELL lines ,GLYCOSYLATION ,HAMSTERS ,RECOMBINANT proteins ,RESEARCH funding ,WESTERN immunoblotting ,METHODOLOGY - Abstract
Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inheritedmetabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84million per year.However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr
- ) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (∼64 and 59 kDa) and secreted (63-69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditionalmethods of screening high-producing recombinant cellsmay represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. [ABSTRACT FROM AUTHOR]- Published
- 2012
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