Overexpression and purification of Dicer and accessory proteins for biochemical and structural studies
| Autor: | Niladri K. Sinha, Brenda L. Bass |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Ribonuclease III
0301 basic medicine Biochemical Phenomena genetic processes Gene Expression Article General Biochemistry Genetics and Molecular Biology 03 medical and health sciences RNA interference Protein purification Sf9 Cells Animals Drosophila Proteins Molecular Biology Gene RNA Double-Stranded chemistry.chemical_classification biology fungi Insect cell culture RNA-Binding Proteins food and beverages Molecular biology Cell biology enzymes and coenzymes (carbohydrates) RNA silencing Drosophila melanogaster 030104 developmental biology Enzyme chemistry Nucleic acid biology.protein Baculoviridae RNA Helicases Dicer |
| Zdroj: | Methods. 126:54-65 |
| ISSN: | 1046-2023 |
| Popis: | The Dicer family of ribonucleases plays a key role in small RNA-based regulatory pathways by generating short dsRNA fragments that modulate expression of endogenous genes, or protect the host from invasive nucleic acids. Beginning with its initial discovery, biochemical characterization of Dicer has provided insight about its catalytic properties. However, a comprehensive understanding of how Dicer’s domains contribute to substrate-specific recognition and catalysis is lacking. One reason for this void is the lack of high-resolution structural information for a metazoan Dicer in the apo- or substrate-bound state. Both biochemical and structural studies are facilitated by large amounts of highly purified, active protein, and Dicer enzymes have historically been recalcitrant to overexpression and purification. Here we describe optimized procedures for the large-scale expression of Dicer in baculovirus-infected insect cells. We then outline a three-step protocol for the purification of large amounts (3–4 mg of Dicer per liter of insect cell culture) of highly purified and active Dicer protein, suitable for biochemical and structural studies. Our methods are general and are extended to enable overexpression, purification and biochemical characterization of accessory dsRNA binding proteins that interact with Dicer and modulate its catalytic activity. |
| Databáze: | OpenAIRE |
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