Good thing takes time

Purification of GST-Fusion Protein
Materials:

PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.3)

PBST: PBS + 0.5% Triton X-100

Glutathione Resin: Glutathione Sepharose 4B (Pharmacia 17-07556-01)

Elution Buffer: 100 mM Tris (pH 8.5-9.0), 20 mM reduced glutathione (aliquot and store @ -20 deg C)

0.5 M IPTG: prepare in H2O and freeze in aliquots

Protease Inhibitors: add to solutions immediately before use


Procedure:


1. Induce expression of GST-fusion protein

* grow 5 ml O/N culture of E. coli strain harboring pGEX plasmid in LB + Carb (50 ug/ml)


* innoculate 500 mls LB+Carb media with 5 mls saturated O/N culture


* grow culture at 30 degrees until mid-log phase (OD600 ~ 0.6 - 0.7)
o typically 2.5-3.5 hours for TG1 host strains
o collect 100 ul cells for analysis and resuspend in 10 ul PBS


* induce fusion protein expression by adding IPTG to 0.1 mM
o grow cells for 3 additional hours at 30 degrees (final OD600 ~ 1.2 - 1.3)
o collect 100 ul cells for analysis and resuspend in 20 ul PBS buffer

* harvest cells by centrifugation 3000 x gav for 8 min (5K rpm in SLA-1500 rotor)

* resuspend cells in 20 mls PBS

* concentrate cells by centrifugation 3000 x g for 8 min and resuspend in final volume of PBS no greater than 5 mls

* transfer to 50 ml conical tube


2. Prepare extract (all work done @ 4 degrees C)

* freeze cells at -80 degrees 1 hr - O/N

* thaw cells and add protease inhibitor cocktail

* lyse cells by sonication: 4 - 6 x 10 seconds with 45 second rests on ice between bursts
o (Fisher 550 Sonic Dismembranator @ setting 4.5 with microtip)
o monitor cell lysis by examination with compound microscope

* add Triton X-100 to final concentration of 1% and rock gently for 20 minutes

* clarify extract by centrifugation
o transfer lysate to centrifuge tube
o wash and collect residual with 5 mls GST buffer and combine with lysate
o spin down insoluble material 10 min @ 8000 x gav (10K rpm in SS-34 rotor)

* transfer supernatant to clean tube
o save 10 ul for analysis

* resupend pellet in 10 mls PBST and save 10 ul for analysis

3. Prepare glutathione resin

* add 1.33 mls 75% glutathione sepharose slurry to 15 ml conical tube ( = 1 ml bed volume)

* spin down resin 2 min @ 500 x g (2K rpm in IEC tabletop centrifuge) and remove supernatant
* wash 2x with cold PBS
* wash 1x with cold PBST

4. Bind GST-fusion protein to GSH-resin

* transfer clarified extract to prepared glutathione resin

* rock gently @ 4 degrees for 2 hours to allow binding

* remove unbound material by centrifugation 2 min @ 500 x g and removal of supernatant

* wash resin 2x with cold PBST

* wash resin 1x with cold PBS

5. Elute bound protein from resin

* transfer resin to empty column and allow resin to settle

* elute protein from resin in 10 x 250 ul fractions

* determine fractions containing protein by Bradford assay
o GST-fusion proteins typically eluted in fractions 3-6

* asses protein quality by SDS-PAGE of protein containing fractions

* pool protein containing fractions and store at -80 degrees


Notes & Misc:

* Often a 70 kD protein co-purifies with the GST-fusion protein. This is likely the chaparonin DnaK and can sometimes be removed by treating the clarified lysate with 2 mM ATP, 10 mM MgSO4 before binding the GST-fusion to the resin.
* Use 25-30 degrees instead of 37 degrees for cultures to improve solubility of GST-fusion proteins
* 1 ml bed volume of resin is reported to bind approx 5-8 mg protein


References:

* GST Gene Fusion System (Pharmacia Biotech)
* solubilization from inclusion bodies: Frangioni, J.V. and B.G. Neel (1993). Anal. Biochem. 210:179-187.



protocol compiled by Chad Rappleye

Aroian Lab Protocols