Production and Purification of Recombinant Glucocerebrosidase in Transgenic Rice Cell Suspension Cultures
| Autor: | Jun-Young Kwon, Dong-Il Kim, Seung-Hoon Kang, Hong-Yeol Choi, Hahn-Sun Jung, Hyung-Jin Nam |
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| Rok vydání: | 2015 |
| Předmět: |
0106 biological sciences
0301 basic medicine Signal peptide Mannose Bioengineering CHO Cells Biology 01 natural sciences Applied Microbiology and Biotechnology Biochemistry Polymerase Chain Reaction law.invention 03 medical and health sciences chemistry.chemical_compound Cricetulus Western blot Suspensions law Cricetinae medicine Animals Humans Secretion Promoter Regions Genetic Molecular Biology Cells Cultured Cell Proliferation chemistry.chemical_classification medicine.diagnostic_test Chinese hamster ovary cell Oryza General Medicine Plants Genetically Modified Recombinant Proteins 030104 developmental biology chemistry Cell culture Recombinant DNA Glucosylceramidase alpha-Amylases Glycoprotein 010606 plant biology & botany Biotechnology |
| Zdroj: | Applied biochemistry and biotechnology. 181(4) |
| ISSN: | 1559-0291 |
| Popis: | Gaucher disease, which is caused by deficiency of glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy. Plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. However, the gliding step for the purification is essential since the produced protein still exists inside cells. In the case of rice-amylase 1A (RAmy1A) secretion signal peptide, GCD protein is secreted outside of cells and simplifies the purification step. Here, an established cell line was confirmed as having fundamental characteristics of growth and production. GCD from transgenic calli was examined by Western blot analysis and compared with that from Chinese hamster ovary (CHO) cells. Calli expressing high levels of GCD were used to establish suspension cell lines. Growth and production characteristics were investigated in suspension cell cultures. Production of GCD in suspension cultures was confirmed upon induction for 12-24 h. The amount of GCD in medium increased until 60-84 h and decreased thereafter. Purification of GCD was performed in three steps (ion exchange, hydrophobic interaction, and size exclusion chromatography) and verified. Purified GCD was able to hydrolyze the synthetic substrate. Thus, a rice expression system could be a suitable alternative to GCD expression in mammalian cells. |
| Databáze: | OpenAIRE |
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