Overexpression and purification of Dicer and accessory proteins for biochemical and structural studies

- An optimized protocol for overexpression of Dicer proteins in insect cells is proposed.
- A three-step purification procedure for obtaining highly-purified, active Dicer is described.
- Overexpression and purification procedures for Loquacious-PD (Loqs-PD) are described.
- Dicer and Loqs-PD are shown to interact in vitro using gel-filtration chromatography.
- Co-expression and purification protocols for Dicer and R2D2 are described.

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.