E63-1 x HIS Purification

updated 07/25/04 - Benjamin Costantino

(For the PDF version, click here.)

Stock Solutions:

LB
100mM IPTG (2.38g IPTG in 100mL ddH₂O; filter sterilize; store -20° C)
Ampicillin or Carbenicilllin (use at 50µg/mL)
Solutions Provided From His-Bind Buffer Kit (Novagen Cat.#69755-1):
8x Binding Buffer
8x Wash Buffer
4x Elute Buffer
4x Strip Buffer
8x Charge Buffer
Other Novagen Products Used:
Pre-packed His-Bind Column (#70971) stored at 4° C
Bug Buster (#70584) stored @ room temperature
Benzonase (#70746) stored at -20° C

Inducing Recombinant Protein Expression and Preparing Cell Lysate

A. Induction
1. Streak Amp-LB plate with freezer stock E63-1xHis BL21 cells and incubate plate overnight.
2. Pick single colony from freshly streaked plate and inoculate 5ml of LB-Amp media. Grow overnight.
3. Inoculate 100ml of LB-Amp media with 1ml of overnight culture.
4. Incubate in shaker at 37° C until OD₆₀₀ = .6 (usually about 3 hours).
5. Remove 500µl of culture as uninduced control. Add IPTG for final concentration of 0.4mM. Incubate for another 2-3 hours. Remove 500ul aliquots at 30min intervals to monitor protein expression over time; process these samples in the section called "SDS-PAGE of Control Samples".

B. Cell Extract Preparation
1. Place flask on ice for 5 minutes, then make 8-10 10ml aliquots in centrifuge tubes.
2. Centrifuge in Hoshizaki centrifuge at 10,000g for 10min. (6,500 RPM JA-20 rotor or equivalent)
3. Decant supernatant and drain off as much liquid as possible by gently tapping upside down on paper towel.
SELECT ONE TUBE FOR PURIFICATION AND STORE THE OTHERS AT -80° C
4. Resuspend pellet in 2.5ml of room temp. BugBuster*/Benzonase (1µl Benzonase per 1ml BugBuster). Gently vortex or pipette to get pellet into solution.
5. Incubate the cell suspension on a shaking platform for 10-20min at room temperature. The solution should not be viscous at the end of the incubation.
6. Remove insoluble cell debris by centrifugation at 16,000g (~16000rpm in Hoshizaki centrifuge) for 20 min.
7. Transfer the supernatant to a fresh 50ml tube. Keep tube on ice until ready to be loaded.

Affinity Chromatography

A. Column Preparation
1. Remove His-Bind Column cap, pour off or pipette all of the storage buffer in the lower chamber and remove plug.
2. Equilibrate the column with 10ml of 1x Binding Buffer. Allow the entire buffer volume to flow through the column.

B. Column Chromatography
1. After Binding Buffer has drained, load the column with the prepared cell extract (should be ~2.5ml of solution).
2. Wash the column with 10ml of 1x Binding Buffer.
3. Wash the column with 10ml of 1x Wash Buffer.
4. Elute the protein from the column with 5ml of 1x Elute buffer. Capture 1ml fractions in 1.5ml Eppendorf tubes.

Processing of Purified Samples

1. Clamp one end of pre-treated tubing with molecular weight cutoff range of 12,000-14,000 (Fisher Scientific Cat. #08-667A, 1cm tubing). Pipet eluted protein into dialysis tubing and clamp the other end shut.
2. Place tubing in a large beaker (3 L) filled with cold Tris storage buffer (10mM Tris-HCl, pH7.5) and stir on stir plate. Make sure stir bar is not hitting the tubing. Dialyse overnight and change storage buffer at least once.
3. Determine the protein concentration by method of choice. For long term storage keep at -80° C; storage at -20° C seems to be alright.

SDS-PAGE of Control Samples

1. Pellet cells from each 500µl aliquot by centrifuging for 5 min. at 6000 RPM. Remove supernatant and resuspend pellet in 50µl 1xLaemmli sample buffer. Store at -20° C until samples are ready to use.
2. Heat samples at 70° C for 5 min. and run 5-10µl on an SDS-polyacrylamide gel (15% for E63-1). Stain with Coomassie Blue 15 - 30 min. Destain with destaining solution to visualize proteins (destaining may take overnight).

NOTES:

* There are no adverse effects of using larger volumes of BugBuster.