Purification of His-Tagged Protein

(TEV protease to cut the tag)

Day 1
• Thaw cells in ice water, add Protein Inhibitor Cocktail (PIC) (0.5-0.25 mM final
concentration)

Sonicate, add more PIC (1 mM)

• Pour cell lysate into centrifuge tubes, balance, spin 17,000 rpm, at 4 ºC, 20-30min.

• Prepare NiNTA resin: use 5 mL of slurry for up to 100 mg of purified protein, spin 5 min
500 rpm, pour off storage solution. Equilibrate with binding buffer (BB): add 10X slurry
volume of BB, spin 5 min 500 rpm, repeat.

• Pour supernatant after centrifugation in to 50mL Falcon tube and add equilibrated Ni resin.

• Rotate the tube 30min at 4 ºC (batch binding).

• Spin the tube at 700-1000 rpm, 5 min, at 4 ºC, keep the pellet (NiNTA resin with bound
protein). Wash resin 3-4 times with 30 mL of wash buffer (WB), each time spinning 5 min
500 rpm.

• Place the resin into a small column, hook up to automatic pump. Continue to wash column
with WB, assaying with Bradford’s Reagent (10 µL from column to 200 µL Reagent) to
assay for presence of protein, until assay baselines (blue color disappears).

• Elute protein using 10 mL or more of elution buffer (EB). Use Bradford’s Reagent to assay
for eluted protein. Continue to elute until assay baselines.

• Add EDTA Na TCEP to eluted protein (bring concentration to 1mM EDTA and 0.5 mM
TCEP ).

• Calculate final yield of protein, use Bradford’s Reagent and check OD 595 nm.

• Check purity of sample: use mass spectrometry or SDS-PAGE electrophoresis.

• If required, remove His-tag: add TEV protease, 30-60 µg protease to 1 mg of protein. Either
leave overnight at 4 ºC in elution buffer solution supplemented with 0.2 M Arginine, or place
in dialysis buffer .

Day 2

• Dialyze in dialysis buffer for 1-2 additional days until TEV cleavage is complete (check
efficiency of TEV cleavage using Masspec analysis or SDS-PAGE electrophoresis).
Days 3-4

• To remove TEV protease, the His tag and uncut protein: pour the dialysed protein in to a
small column containing 3-5 mL of NiNTA resin that was equilibrated by BB. Make sure to
keep the flowthrough: contains protein with His tag removed. Use Bradford’s Reagent to
assay flowthrough for protein. If necessary, add WB or EB for elution, which indicates TEV
cleavage did not reach completion or ability of the protein to bind Ni-resin with the help of
own Histidines.

• Dialyze against precrystallization buffer.

• If cleavage of His tag was totally unsuccessful, upon next purification dialyze protein against
precrystallization buffer directly after elution from first NiNTA column.

• Concentrate protein to final volume of 200-500 µL, attempt to reach final concentration of at
least 10 mg/mL if total yield of protein allows.

• Filter sample through a 0.22 µm PVDF membrane and calculate amount of protein using
Branford’s Reagent (OD 595).

• Use fresh protein for crystallization experiments.

• Aliquot the concentrated protein into PCR tubes, flash freeze them in liquid nitrogen and
store at –70 ºC.

• For purification of Se-Met derivatized proteins and TEV protease: all buffers should
include 0.5 mM TCEP.