Berglund:Protease1

Berglund Lab: Protease Purification

For information see: Danielle Cass Lab Book 1/5/04 (Dates 7/15-7/20)

Vector: Pgex4Tl (BamH1, EcoR1 insert sites)

Transform plasmid into BL21 competent cells (Day 1).

Overnight Culture (Day 2)

1. Start a 50mL culture of 2xYT with 100μg/mL Amp (50μL 1000x Amp stock).
2. Pick one colony from BL21 transformation plate.
3. Shake overnight at 37°C.

Large Scale Culture (Day 3)

1. Dilute overnight culture to 1L with 2xYT + 100μg/mL Amp (1mL Amp stock).
2. Shake at 37°C until OD₆₀₀ = 1.2
3. Add IPTG to 0.25mM (500μL of 0.5M IPTG per 1L Culture)
4. Shake at 37°C for 3 hours.
5. Spin down cells at 6000 RPM for 15 min and store in -20°C overnight (-80°C if storing longer).

Cell Lysis (Day 4)

1. Resuspend cells in up to 45mL Lysis Buffer with 2mM fresh DTT.
2. Sonicate cells in glass beaker, on ice.
   • Tune sonicator according to directions.
   • Sonicate at power setting 7, 3x30sec.
3. Spin down in JA-17 12,000 RPM for 30min.
4. Save supernatant, discard pellet.

GST Beads

1. Wash 5ml packed GST beads (~7.5m-10mL suspended).
   • Spin beads down at 1000 RPM for 3 min.
   • Remove supernatant.
   • Add 45mL Lysis Buffer, mix.
   • Spin down, remove supernatant.
   • Repeat 3x from adding Lysis Buffer.
2. Add supernatant from cell lysis to washed GST beads.
3. Rotate 1 hour at 4°C.
4. Wash beads 3x with 45mL Lysis Buffer.
5. Elute GST-Protein off of beads.
   • Add 10mL Elution Buffer
   • Shake at RT l0 min.
   • Spin down beads at 1000 RPM, 3 min.
   • Remove AND SAVE elution.
   • Repeat 3x

Dialysis

1. Place GST-eluted Protease into 15kd MWCO dialysis tubing.
2. Dialyze in 1L Dialysis Buffer for 4 hours at 4°C, change buffer and dialyze overnight.

Usage of Protease (Day 6)

1. Take protein concentration. YOU MUST USE A SPECTROPHOTOMETER for quantitation. The Bradford will give you a 10 fold less number.
2. Aliquot out ~0.04 μmol protease (~100μL of 400μM).
   • Abs coefficient = 5120 M^-1cm^-1
3. Store at -20°C.

Buffers

Lysis Buffer (500mL)

• 25mM Tris pH 7.5 (12.5mL of 1M)
• 300mM NaCl (30mL of 5M)
• 1mM EDTA (1mL of 0.5M)
• 2mM DTT (0.5mL of 2M) (add after filtering with a .22μm vacuum filter)

Elution buffer (500mL)

• 50mM Tris pH 8 (15ml of 1M)
• 3.08mg/ml reduced glutathione (add fresh)

Dialysis Buffer (1L)

• 150mM NaCl (30mL of 5M)
• 10mM EDTA (20mL of 0.5M)
• 1mM DTT (0.5mL of 2M)
• 50mM Tris pH 8 (50mL of 1M)
• 20% Glycerol (200mL of 100%)

Suggested Cutting Conditions

• 150mM NaCl
• 50mM Tris-HCl pH 7.0
• 1mM EDTA
• 1mM DTT

At 4°C for 16 hours.