Production and Characterization of Recombinant Mouse Brain-Derived Neurotrophic Factor and Rat Neurotrophin-3 Expressed in Insect Cells
| Autor: | Stephen P. Trusko, Meyer Sl, James D. Hirsch, Richard W. Scott, Knight E, Diane M. Lang, Forbes Me |
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| Rok vydání: | 2008 |
| Předmět: |
Insecta
Genetic Vectors Molecular Sequence Data Oligonucleotides Nerve Tissue Proteins Neurotrophin-3 Tropomyosin receptor kinase B Nervous System Biochemistry Cell Line law.invention Mice Cellular and Molecular Neuroscience Neurotrophin 3 Neurotrophic factors law Gene expression Animals Nerve Growth Factors Neurons Brain-derived neurotrophic factor Base Sequence biology Brain-Derived Neurotrophic Factor Intracellular Membranes Molecular biology Recombinant Proteins Rats Nerve growth factor Solubility nervous system Cell culture biology.protein Recombinant DNA Baculoviridae |
| Zdroj: | Journal of Neurochemistry. 62:825-833 |
| ISSN: | 1471-4159 0022-3042 |
| DOI: | 10.1046/j.1471-4159.1994.62030825.x |
| Popis: | Bioactive brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were produced using the baculovirus expression system and purified to homogeneity using ion-exchange and reversed-phase chromatography. Yields of purified neurotrophin-3 (300-500 micrograms/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20-50 micrograms/L). Improved production of BDNF (150-200 micrograms/L) was achieved by expressing BDNF from a chimeric prepro-NGF/mature BDNF construct using the Trichoplusia ni insect cell line. Tn-5B1-4. Examination of the distribution of BDNF protein from both the non-chimeric prepro-BDNF and the chimeric prepro-NGF/mature BDNF viruses in Sf-21- and Tn-5B1-4-infected cells suggests a specific deficiency in the Tn-5B1-4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N-terminal amino acid sequencing of purified recombinant BDNF and neurotrophin-3 demonstrated that the insect cells processed their precursors to the correct N-terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS. |
| Databáze: | OpenAIRE |
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