Overexpression and purification of Dicer and accessory proteins for biochemical and structural studies.
Methods
; 126: 54-65, 2017 08 15.
Article
em En
| MEDLINE
| ID: mdl-28723582
ABSTRACT
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 (3-4mg 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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA de Cadeia Dupla
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Proteínas de Ligação a RNA
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RNA Helicases
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Proteínas de Drosophila
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Ribonuclease III
Limite:
Animals
Idioma:
En
Revista:
Methods
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2017
Tipo de documento:
Article