Human Argonaute-2 expression/purification protocol
Leemor Joshua-Tor’s Laboratory, CSHL
Expression vector: pFl (MultiBAC expression system) with Strep-Strep-Sumo-TEV-hAgo2.
The pFl vector also expresses YFP under the p10 promoter to monitor expression level. The use of the MultiBac expression system in which proteases are disrupted could be particularly important in this case.
Note: Low YFP expression (<90-95%) will result in very low yields and no RNA-free fraction!
SF9/Hi5 were infected for 72 hours.
Expression yields: Yields for hAgo2 bound to endogenous SF9/Hi5 RNA are ~ 1-2mg/L of cells (after strep purification). RNA free hAgo2 is usually no more than 10-15% of the total hAgo2 expression. In order to obtain significant amounts of RNA-free hAgo2, it is recommended to express >5L of SF9/Hi5 cells. The cation exchange chromatogram in Elkayam et. al (Cell 2012) is from a purification preparation of 8L of SF9 cells.
1. SF9/Hi5 cells are re-suspended in the following buffer: KCl 100mM, Tris 50mM, DTT 5mM, and PI cocktail (tablets from Roche, EDTA free or home made cocktail) flash freeze in liquid N2 and store in -80°C.
Thaw the frozen cells inside water beaker at RT.
Add KCl to a final concentration of 400mM.
Sonicate for 1 min with 1 sec on 5 sec off at 70% amplitude in an ice water bath.
Add PEI to a final concentration of 0.2% (V/V), mix by inverting a few times, do not vortex!!!
Immediately spin at 35K for 45min using ultracentrifuge (rotor Ti 45).
1. wash/binding buffer: Tris 50mM pH 8.0, KCl 100 mM, 5 mM DTT.
2. High salt wash buffer: wash buffer with 1.0M KCl.
3. Elution buffer: wash buffer with 5 mM desthiobiotin.
- Mix the Strep-Tactin with the supernatant incubate for 30 min
- Apply to the column and let it flow by gravitation
- Wash with 1 CV X10 (buffer 1)
- Wash with 1CV X3 (buffer 2)
- Wash with 1CV X5 (buffer 1)
- Elute with 1C X5 (buffer 3) with 5 ml fractions
Run the fractions on a gel and combine them based on the gel.
Incubate with TEV protease at 1:50-100 overnight at 4°C.
Mono S column
Dilute the sample with 50mM Tris pH=8.0 to lower the KCl conc. to 50 mM (the elution buffer from the Strep is 100mM KCl)
- Filter the sample using a syringe filter.
- Equilibrate the Mono S column with Tris 50mM pH 8.0, KCl 50 mM, 5 mM DTT, until both UV and conductivity are stable (~10CV).
- Load the sample on the column (sample pump or super loop).
- Wash the column for 10-15CV with Tris 50mM pH 8.0, KCl 50 mM, 5 mM DTT.
- Elute using a linear gradient of Tris 50mM pH 8.0, KCl 1000 mM, 5 mM DTT over 30 CV (this step is crucial to determine what step gradient to use next time, the KCL concentration can change slightly between different column between different cation exchangers.
- Verify the RNA free Ago fraction by measuring 260/280 nM ratio and by running a urea gel after RNA Trizol extraction.
- Concentrate the protein to 5-10 mg/ml using Amicon Ultra 50kDa cutoff.
Equilibrate Superdex 200 10/300 with Tris 20mM pH 8.0, NaCl 200 mM, 5 mM DTT
Inject the concentrated sample (up to 1 ml at 5 mg/ml).
The elution volume of both the bound and bound protein should be at 14.5-15ml.