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

Niladri K. Sinha_ and Brenda L. Bass_
Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA

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.

Related Application Notes

Optimum Growth™ Protocol for Insect Cells

... Sf9 and Sf21 insect cells are cell lines commonly used for expression of recombinant proteins using baculovirus sys...

See Application Note

Simplified Sample Prep for Open Access SEC-HPLC Detection using the Thomson Filter Vials

Routine analysis of samples containing cell culture media and/or supernatant by HPLC can lead to performance issues and...

See Application Note

Comparison of the Rapid Clear® Cap 3000 (patented) vs GE Capsule Filters for clarifying monospecific IgG

Two methods to clarify 10L of post transfected IgG harvested from culture are compared. The culture was harvested on da...

See Application Note

Improved DNA Protocol for E.coli with Plasmid+® Media

Plasmid+® liquid media is an enriched media specifically designed for plasmid DNA production. Plasmid+® supports much h...

See Application Note

Title

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

Description

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....

PDF Download

Products Used

24-Well Filter Plate | ~9mL

Square Well, Long Drip | 0.2µm Rapid Clear Filter

pn#921546

24-Well Filter Plate | 10.8mL

Square Well, Long Drip | 25µm Polypropylene Filter

pn#921550

24-Well Plate | 10.4mL

Square Well, Round Bottom | Individually Wrapped | Sterile

pn#931565-G-1X

Related Products

© 2021 Thomson |
Solutions At Work™

LinkedIn