Purification of recombinant sBRF M166L

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

last modified 10/9/00

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 (50μg/ml)(and Kan (30μg/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)

30mM Tris 8.0 0.54 g Tris

300mM NaCl2.63 g NaCl

6.0M Gu-HCl86 g Guanidine-HCl

10mM Imidazole 0.1 g Imidazole

Wash Buffer,pH8.0 (200 ml)

30 mM Tris 8.0 0.73 g Tris

300mM NaCl3.5 g NaCl

8.0M Urea 96.2 g Urea

10mM Imidazole 0.136 g Imidazole

H₂O to 200 ml final

Elution Buffer,pH 8.0 (50 ml)

20mM Tris 8.0 0.12 g Tris

300mM NaCl 0.87 g NaCl

8.0M Urea 24 g Urea

100mM Imidazole 0.33 g Imidazole

H₂O to 50 ml final

Renaturation of BRF protein

Thaw Ni-NTA purified samples.Dilute the sample to a concentration of 0.25 to 0.5mg/ml in the elution buffer.To the BRF sample,add 10% Brij58 detergent to a final concentration of 0.1% Brij,add DTT to 5 mM final concentration,add ZnS04 to a concentration of 20 micromolar,and add all protease inhibitors to 1X concentration.For each dialysis step,use a buffer to sample ratio of 40:1.Dialyze 4 times for four hours each time into decreasing amounts of Urea: 4M,then 2M,then 0.75M,and then 0M Urea.Spin renatured protein in the SS34 rotor for 10 min at 10K rpm.If a large pellet is detected,analyze the samples by SDS PAGE before proceeding with the purification to confirm that the renatured BRF is soluble.

--------------------------------------------------------------------------------

BRF Renaturation Buffer (1.5 liters)

10% glycerol 150 ml glycerol

20 mM HEPES 7.9 7.14 g HEPES

150 mM KCl16.8 g KCl

5 mM MgCl₂ 7.5 ml 1 M MgCl₂

1 mM EDTA 8 ml 0.25 M EDTA

20 micromolar ZnSO₄ 0.3 ml 0.1 M ZnSO₄

0.1% Brij 58 1.5 g Brij 58

H₂O to a final volume of 1.5 liters

Add protease inhibitors and DTT to 5 mM just prior to dialysis.

Protease inhibitors for BRF renaturation:

0.1 M PMSF (100x)

16 mg/ml Ethanol

Store at -20 degrees

Benzamidine (100X)

31 mg/ml H₂O.

Store frozen at -20 degrees

Leupeptin (500X)

0.15 mg/ml Ethanol.

Store at -70 degrees for less than 6 months

Pepstatin (200X)

0.28 mg/ml methanol

Store at -20 degrees.

Chymostatin (2,500X)

5mg/ml DMSO

Store frozen at -20 degrees

Purification of BRF using SOURCE 15Q Ion Exchange Chromatography

Thaw the renatured BRF (in 150 mM KCl)and dilute with buffer B +0 KCl to a final KCl concentration of 50 mM.Filter the diluted sample through a 0.22 micron syringe filter before loading.

Load the filtered sample to a 0.5 ml SOURCE 15 Q column equilibrated in B + 50 mM KCl at a flow rate of 0.5 ml/min.

Wash the column with 6 column volumes of B + 50 mM KCl.

Elute with a 50 column volume gradient from 50 mM KCl to 550 mM KCl.Collect 0.5 ml fractions.BRF elutes in 2-3 fractions after about 1/3 of the gradient has completed at a conductivity of about 12 mS/cm-16mS/cm on the ATKA instrument.Expect 0.5- 1 mg of highly purified BRF protein from 10ml of renatured and diluted BRF.

Wash the column with 5 column volumes of B + 1.0 M KCl and 5 column volumes of 20% ETOH to clean.If the column pressure exceeds pressure limits and the ETOH cleaning does not cure the problem,follow the more extensive cleaning procedure recommended by Pharmacia.

--------------------------------------------------------------------------------

Buffers for Ion Exchange Chromatography

B + 0 NaCl (500 ml)

30 mM Tris 8.0 1.82 g Tris

1 mM EDTA 2 ml 0.25 M EDTA

10% glycerol 50 ml glycerol

H₂O to a final volume of 500 ml

B + 1 M NaCl (500 ml)

30 mM Tris 8.0 1.82 g Tris

1 mM EDTA 2 ml 0.25 M EDTA

10% glycerol 50 ml glycerol

1 M KCl37.3 g KCl

H₂O to a final volume of 500 ml

Add DTT to 1 mM and protease inhibitors to 1x before use.Filter buffers through 0.22 micron filter before using on FPLC.

Pefablock (Boehringer)(1000x)

100 mg/ml H₂O stock store at -20 deg.

Leupeptin (500X)

0.15 mg/ml Ethanol.

Store at -70 degrees for less than 6 months

Pepstatin (200X)

0.28 mg/ml methanol

Store at -20 degrees.

Back to Hahn Lab Methods Index