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.
 * 3) *Tune sonicator according to directions.
 * 4) *Sonicate at power setting 7, 3x30sec.
 * 5) Spin down in JA-17 12,000 RPM for 30min.
 * 6) Save supernatant, discard pellet.

GST Beads

 * 1) Wash 5ml packed GST beads (~7.5m-10mL suspended).
 * 2) *Spin beads down at 1000 RPM for 3 min.
 * 3) *Remove supernatant.
 * 4) *Add 45mL Lysis Buffer, mix.
 * 5) *Spin down, remove supernatant.
 * 6) *Repeat 3x from adding Lysis Buffer.
 * 7) Add supernatant from cell lysis to washed GST beads.
 * 8) Rotate 1 hour at 4°C.
 * 9) Wash beads 3x with 45mL Lysis Buffer.
 * 10) Elute GST-Protein off of beads.
 * 11) *Add 10mL Elution Buffer
 * 12) *Shake at RT l0 min.
 * 13) *Spin down beads at 1000 RPM, 3 min.
 * 14) *Remove AND SAVE elution.
 * 15) *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.
 * 2) Aliquot out ~0.04 μmol protease (~100μL of 400μM).
 * 3) *Abs coefficient = 5120 M-1cm-1
 * 4) Store at -20°C.

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
At 4°C for 16 hours.
 * 150mM NaCl
 * 50mM Tris-HCl pH 7.0
 * 1mM EDTA
 * 1mM DTT