Purification of recombinant sBRF M166L

J. Movius, K, Coachman, and S. Hahn (Hahn Lab)

Induction of BRF in bacteria and purification on Ni-NTA agarose

Transform BRF plasmid into strain BL21 DE3 (pLysS) or JM25 (DE3 containing the Arg tRNA over expression plasmid under lac iQ control Kanomycin resistant)

Inoculate 6 ml of a saturated overnight culture of the transformed bacteria into 2X 1 Liter of YT Amp (50 ug/ml) (and Kan (30 ug/ml) if using strain JM25) (adding 1-2 drops of antifoam A before autoclaving the YT will decrease the foam in the culture during growth). Incubate at 30 deg till the culture reaches OD600 ~ 0.6. Remove a small 1 ml sample for an uninduced control, and incubate this small sample on the tube roller for an additional 4 hrs.

Induce the large culture with 0.4 mM IPTG (4 ml of 0.1 M IPTG per 1000 ml) and grow at 30 deg. for four hours. Take a small sample of the induced and uninduced cultures and save for analysis of whole cell protein by SDS PAGE.

Harvest cells by centrifugation (4-6g). Resuspend the cell pellet in 160 ml Lysis buffer. The cells can be frozen at this point in liquid N2 and stored at -70 deg.

It is usually a good idea to analyze the BRF induction by analysis of total cell protein before proceeding.

Thaw the lysed cells at room temp. Sonicate 2 times for 30 sec. each time using the large sonicator tip. Alternatively, the cells can be run once through the microfluidizer.

Centrifuge the crude lysate in the GSA rotor for 30 min at 10K RPM.

During the above GSA spin, prepare the Ni-NTA resin for BRF binding. Transfer 12 mls of Ni-NTA affinity bead slurry (50% beads; 6 ml of beads total) to a 50ml conical tube and centrifuge 3min at 3K RPM in the GPR centrifuge.

Equilibrate the beads 2X with Lysis buffer by resuspension and centrifugation.

Add the equilibrated beads to the clarified extract and place on a room temp. shaker platform for 45 min.

Centrifuge the bead-extract mix in the GSA rotor for 5 min at 5K RPM and carefully remove the supernatant (the pellet of beads will not be very firm so it seems best to remove as much supernatant as possible with a pipette and don’t worry if not all the sup. can be removed). Save this supernatant in case there is a problem with the BRF binding.

Resuspend the beads with a small amount of Lysis buffer and transfer to a 50 ml conical tube. Use extra lysis buffer to wash the GSA bottle to remove all beads. Spin in the GRP rotor and carefully remove sup. by dumping (the pellets will not be very firm so it is OK if not all the sup. can be removed. Wash the beads 5X with ~35 ml Wash Buffer.

After the final wash, transfer the beads to 15ml conical tubes with Wash Buffer. Centrifuge the beads and try to remove all of the supernatant with a Pasteur pipette leaving the beads semi-dry.

Add a volume of Elution Buffer which is equal to the volume of the beads (6 ml) and place on a roller at RT for 20 min. Centrifuge the beads in the GPR and remove the supernatant and save; this is the first protein elution. Repeat the elution and freeze samples. Analyze recovery by SDS PAGE and/or protein assay. Typical recovery is 2 to 6 mg BRF protein.

Lysis/Binding buffer, pH 8.0 (150 ml)

Wash Buffer, pH8.0 (200 ml)

Elution Buffer, pH 8.0 (50 ml)