Isolation by anion-exchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells
| Autor: | E. Mortensen, Thomas Dyrberg, Kim Ry Hejnaes, Michael O. Marshall, A. J. Moody, Esper Boel, F. S. Larsen, I. Svendsen |
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| Rok vydání: | 1995 |
| Předmět: |
Adult
Carboxy-lyases Adolescent Endocrinology Diabetes and Metabolism Molecular Sequence Data Glutamate decarboxylase Sf9 Spodoptera Gene Expression Regulation Enzymologic law.invention Islets of Langerhans Affinity chromatography law Internal Medicine Animals Humans Child Autoantibodies DNA Primers chemistry.chemical_classification Base Sequence biology Glutamate Decarboxylase Infant Newborn Infant Middle Aged Chromatography Ion Exchange Recombinant Proteins Diabetes Mellitus Type 1 Enzyme Biochemistry chemistry Cell culture Child Preschool biology.protein Recombinant DNA Antibody Baculoviridae |
| Zdroj: | Diabetologia. 38:14-23 |
| ISSN: | 1432-0428 0012-186X |
| DOI: | 10.1007/bf02369348 |
| Popis: | The enzymel-glutamic acid decarboxylase is a major autoantigen of the beta cell. Autoantibodies against this enzyme are observed before the onset of insulin-dependent diabetes mellitus (IDDM) in man and may be of predictive value. There is evidence that this enzyme is involved in the development of autoimmune diabetes in animals. In order to facilitate the investigation of the role ofl-glutamine acid decarboxylase in IDDM, we expressed the 65 kDa isoform of human isletl-glutamic acid decarboxylase in insect cells using a baculovirus-based vector. The material was expressed at high levels (up to 50 mg/l of cells). Partially purified metabolically labelledl-glutamic acid decarboxylase bound to immunoglobulins in the sera from 20 of 49 subjects with newly-diagnosed IDDM. The enzyme was isolated in high yields (up to 26 mg/l cell culture) with fully maintained enzymatic activity by either ion-exchange chromatography or immunoaffinity chromatography. Purifiedl-glutamic acid decarboxylase inhibited the binding of radioactivel-glutamic acid decarboxylase, prepared by in vitro translation of mRNA, to immunoglobulins in the sera of subjects with IDDM. Recombinant human isletl-glutamic acid decarboxylase, isolated from Sf9 cells, is a suitable material for the large scale investigation of the utility of this enzyme in the prediction and prevention of autoimmune diabetes. [Diabetologia (1995) 38: 14–23 |
| Databáze: | OpenAIRE |
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