title: Generation of a Chinese hamster ovary cell line producing recombinant human glucocerebrosidase.
abstract: Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic 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 $ 84 million 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, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Avenue Vital Brasil 1500, 05503-900 São Paulo, Brazil.
authors.forename: Juliana Branco
authors.initials: JB
authors.lastname: Novo
authors.forename: Ligia
authors.initials: L
authors.lastname: Morganti
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Moro
authors.forename: Adriana Franco
authors.initials: AF
authors.lastname: Paes Leme
authors.forename: Solange Maria de Toledo
authors.initials: SM
authors.lastname: Serrano
authors.forename: Isaias
authors.initials: I
authors.lastname: Raw
authors.forename: Paulo Lee
authors.initials: PL
authors.lastname: Ho
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000166:cytology* / Q000502:physiology*; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005962:Glucosylceramidase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D011994:Recombinant Proteins; D005962:Glucosylceramidase