Protein Purification using Terrific Brot

Protein Purification using Terrific Broth and SUMO-tag

Media:

Terrific Broth Potassium Phosphate Solution

12g Bacto tryptone 2.31g KH2PO4

24g Bacto yeast extract 12.54g K2HPO4

4ml glycerol Adjust volume to 100ml.

Adjust to 900ml. Autoclave. Add 100ml sterile potassium phosphate solution.

Buffers:

Lysis/Binding Buffer (L/B)

20mM Tris Cl pH (8.0)

300mM NaCl

10mM Imidazole

Wash Buffer

20mM Tris Cl pH (8.0)

300mM NaCl

30mM Imidazole

Elution Buffer

20mM Tris Cl pH (8.0)

300mM NaCl

50, 100, 250, and 500 mM Imidazole

Inoculate single colony of E. coli strain cells containing the expression plasmid (vector pTB145) into 25ml culture of LB amp100 cat50. Incubate overnight at 37ºC with shaking.

Inoculate 5ml of overnight culture into 1500ml Terrific Broth (TB) or Luria Broth (LB) amp100.

Grow cells at 37ºC with shaking until OD600 = ~0.6.

Add 750ml 1M IPTG to each flask. (Final Conc. = 0.5mM)

Transfer flasks to 25 ºC and incubate overnight with shaking. *Put rotor at 4 ºC to cool for use in the morning.

Collect 1ml of overnight culture – Sample A. Measure OD600.

Harvest cells via centrifugation. Rotor GS-3 and 500mL bottles @ 6100 rpm for 10 min. @ 4 ºC.

Pour off supernatant. Resuspend in 15ml Lysis/Binding (L/B) Buffer. Transfer to 50 ml conical. Spin at 5000 rpm for 7 min. Remove supernatant.

Resuspend pellet in18ml Lysis/Binding buffer. Add 10ml Nuclease, 10uL protease solution.

Sonicate the cell suspension in a beaker kept on ice.

Harvest 100ml of lysed cells– Sample B

Spin 5 min @ 5000 rpm to pellet most of debris.

Pour supernatant into 40ml centrifugation tube. Balance with another tube.

Centrifuge at 10000 rpm, 4 ºC, with vacuum for 60 min. to clear the lysate.

Equilibrate Ni-NTA beads by gently swirling the bottle to resuspend them. Transfer 1 ml to a 15 ml conical tube. (You may need to cut the end off of blue tip)

Spin at 4000 RPM for about 3 min. Remove sup with pipette tip. Add 1 ml L/B buffer and gently resuspend. Repeat. Keep beads at 4 ºC until ready to use.

Harvest 100ml cleared lysate. Sample C

Pour remaining lysate into 50ml conical. Add equilibrated beads.

Rotate end-over-end for 1 hr at 4ºC.

Apply to column (may require 2 additions) and allow flow to pack. Collect flow through in 50ml conical. Label and save. (Don’t be greedy. Try to not let the fluid level reach the top of the bead bed. You don’t want the beads to dry out.)

Wash beads with 2ml Wash Buffer, rinsing down sides of column. Collect in 2ml tube.

Wash 3x with 2ml Wash buffer, collecting each wash in a separate 2 ml tube.

Step Elute. Collect each elution separately. Use a pasteur pipette to resuspend the bead bed at the beginning of each concentration increase.

2x 0.25ml 50mM Imidazole Elution Buffer

3x 0.25ml 100mM Imidazole Elution Buffer

3x 0.25ml 250mM Imidazole Elution Buffer

2x 0.25ml 500mM Imidazole Elution Buffer

Store flow-thru, washes and elutions at 4ºC. Freeze pelleted samples.

Run all 10ml of samples on a 12% SDS-Polyacrylamide gel. (For small gels, 150V for ~1.5 hr). Stain with Coomassie stain and destain with Methanol:Acetic Acid solution.

Pool elutions with the brightest and cleanest bands. Dialyze into appropriate buffer.

SUMO-fusions: 50mM Tris (pH 8.0)

150mM NaCl

10% glycerol

1mM DTT

Freeze @ -80ºC

SUMO-protease 50mM Tris (pH 8.0)

200mM NaCl

10% glycerol

1mM DTT

THEN into: 25mM Tris (pH 8.0)

200mM NaCl

50% glycerol

0.5% NP-40

0.5mM DTT.

Freeze @ -80ºC