May 13, 2026
Purification of MBP-fused HTLV-1 Tax (Miura et al. 2026)
This protocol is a draft, published without a DOI.
- Michi Miura1
- 1tmpu
Protocol Citation: Michi Miura 2026. Purification of MBP-fused HTLV-1 Tax (Miura et al. 2026). protocols.io https://dx.doi.org/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 07, 2026
Last Modified: May 13, 2026
Protocol Integer ID: 316480
Keywords: purification of mbp, purification, tax
Funders Acknowledgements:
JSPS
Grant ID: 26K10051
Abstract
This step-by-step protocol (on 27 March 2026, just for my record) is to complement the method described in Miura et al. 2026 to purify MBP-Tax-His and MBP-His-Tax.
Bacteria lysis
# Lyse the bacteria and clear the lysate.
Prepare 50 mL Lysis buffer:
50 mM Tris-HCl pH 8.0
400 mM NaCl
10% Glycerol
0.1% Triton X-100
1 mM TCEP
Dissolve one tablet of EDTA-free protease inhibitor cocktail (Roche, 04693159001).
Resuspend frozen bacterial pellet (harvested from ~50 mL overnight bacterial culture) in 10 mL Lysis buffer.
Sonicate the bacteria to disrupt the cell wall.
Centrifuge the lysate at 2,100xg, 10 min, 4°C. Collect supernatant.
Centrifuge the supernatant again at 16,000xg, 10 min, 4°C. Collect supernatant.
Purification using starch slurry for MBP
# Purify MBP-fused Tax using corn starch slurry (Kobashigawa et al. 2021) that captures the MBP moiety.
Prepare corn starch slurry according to Kobashigawa et al. 2021. Below is what we normally do for slightly large-scale preparation:
- Take ~1 g corn starch powder in a 50 mL conical tube and resuspend in ~20 mL Milli-Q water.
- Place the conical tube in a ~85
°C water bath and incubate for 10 min. Vortex briefly every minute to keep the slurry suspended. - Let the slurry cool down.
- Wash the slurry with plenty of Milli-Q water (requires 2,600xg for 2 min to harvest).
- Make 50% slurry with water, and store it in the fridge until use.
Combine the ~10 mL supernatant with the slurry (up to ~2 mL gel volume) equilibrated with Lysis buffer minus protease inhibitors in a 15 mL conical tube. Incubate for ~1.5 hours at 4°C.
Centrifuge to collect the slurry.
Wash the slurry with ~10 ml Lysis buffer minus protease inhibitors.
Incubate the slurry with ~2 ml Lysis buffer containing 100 mM maltose for ~1 hour at 4°C to elute.
Centrifuge to collect the eluate.
HRV 3C treatment
# Cleave the recombinant protein between MBP and Tax.
Add ~4.5 μg/ml HRV 3C and incubate overnight at 4°C.
Purification of His-tagged Tax using Ni-NTA
# Isolate histidine-tagged Tax from the MBP moiety using Ni-NTA.
Dilute the HRV 3C-treated sample by 2-fold with 50 mM Tris-HCl pH 8.0 to reduce the concentration of NaCl and maltose.
Combine the sample with the Ni-NTA resin (~0.2 mL gel volume) that has been washed with PBS/0.01% Tween20. (PBS/0.01% Tween 20 can be replaced with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.01% Tween 20 from this step onwards.) Incubate for ~1.5 hours at 4°C.
Centrifuge to remove the unbound.
Wash the resin with PBS/0.01% Tween 20 containing 20 mM imidazole.
Incubate the resin with 200 μL PBS/0.01% Tween 20 containing 400 mM imidazole for ~1 hour at 4°C to elute. (400 mM is an overkill - 200 mM also works.)
Centrifuge to collect the eluate.
Desalting
# Remove imidazole using a centrifugal filter.