Aliquot of Cell Pellet after Induction

The idea is to aliquot cells after induction, and keep at  -80ºC enough cell pellet samples for optimization of small scale
purification procedure and further scale-up.  Once you set up the best purification conditions at low scale, you can scale-up
the procedure.

Equilibration of Glutathione Agarose

Place 20ul beads (40ul suspension) of glutathione agarose in 1.5ml plastic tube.

Wash with 2 x 1.5ml H₂O and 2x 1.5ml lysis buffer (washing: mix, spin 3min 3500rpm, discharge supernatant).
 

Protein Extraction

Resuspend pellet of 10ml cell culture in 1ml lysis buffer (or 100ml bacterial culture for very low expression level).

Suggested Lysis buffer : 140mM NaCl; 2.7mM KCL; 10mM Na₂HPO₄; 1.8mM KH₂PO_4; pH 7.3 (PBS)
                                    optional 0.02% NaN₃ (azide)
                                    optional protease inhibitors

Optional additives to the lysis buffer
a) 1mM PMSF or protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma)
b) Dnase 100U/ml or 25-50ug/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of 10mMMgCl₂.
c) Lysozime 0.2mg/ml. Incubate 10min 4°C.
d) ßME, DTT or DTE  up to 10mM for proteins with many cysteines.
e) 0.1-2% Triton X-100, NP40; or any other detergent that do not affect the biological activity of your protein.

Sonicate in ice bucket 3 x 10sec or more if the cells are not completely disrupted (Lysis is complete when the cloudy cell suspension becomes
translucent. Avoid protein denaturation by frothing).

Spin 5min 13000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet). Use supernatant for next
step. Keep sample of 40ul of supernatant for PAGE-SDS: soluble proteins

Resuspend pellet in another 1ml lysis buffer and keep sample of 40ul for PAGE-SDS: insoluble proteins, or unlysed cells.
 

Protein Purification

1) Mix supernatant of last step gently with the equilibrated resin 60 min at 4°C.

2) Spin 3min 3500rpm 4°C. Discharge supernatant and keep sample of 40ul for PAGE-SDS: unbound proteins (this material could be use
again in case of overloading).

3) Wash beads with 2x1.5ml PBS (washing: mix, spin 3min 3500rpm, keep supernatant aside). Keep sample of 40ul for PAGE-SDS of each washing.

4) Elute recombinant protein with 3 x 50ul elution buffer: 50mM TrisHCl pH8.0 10mM reduce glutathione (mix gently, incubate at RT for 5min for each
elution, spin 3min 3500rpm , keep supernatant aside). Keep sample of 40ul for PAGE-SDS of each elution.

5) Resuspend beads in 50ul H₂O + 20ul 5x sample buffer. Mix and spin. Keep sample of 40ul for PAGE-SDS: protein not eluted  (or SDS
extracted beads).

6) Run on PAGE-SDS: crude supernatant; resuspended pellet; unbound, washings, elutions, and SDS extracted beads. MW of GST alone: 29000 

Large Scale

Cleaning and Storage of Glutathione Sepharose High Performance
According ot Amersham Biosciences Instructions

If the medium appears to be losing binding capacity, it may be due to an accumulation of precipitate, denatured or nonspecifically
bound proteins.
Removal of precipitated or denatured substances:
• Wash with 2 column volumes of 6 M guanidine hydrochloride, immediately followed by 5 column volumes of PBS, pH 7.3.
Removal of hydrophobically bound substances:
• Wash with 3-4 column volumns of 70% ethanol or 2 column volumes of 1% Triton™ X-100, immediately followed by 5 column volumes of H₂O and then
5 column volumes of PBS, pH 7.3.
Resin Storage
• Wash resin with 5 column volumes of H₂O and then store the packed column or resin at 4°C in 20% ethanol.

Analysis of results - Troubleshooting

Expect over-expressed protein to be found only in the crude supernatant and in the elution of the Glutathione agarose.

If most of the protein remains insoluble after extraction, try
a) To change lysis buffer by adding ßME, DTT, glycerol, detergents or more NaCl. If only part of it is insoluble,
b) Or re-extract pellet with more buffer,
c) Or use more lysis buffer during extraction,
d) Or perform a more intensive sonication,
e) Or incubate with lysozyme before sonication.
f) Or try the denaturating protocol extraction (4-6 M guanidine-HCl; 4-8 M urea; alkaline pH>9.0; 0.5-2% Triton X-100; 0.5-2% N-lauroylsarcosine or
other detergents)

If protein does not bind to the Glutathione agarose, there are several options to choose:
a) Check the Glutathione agarose: binding of a cell sonicate containing only GST
b) If only partially bound, use more resin, or bind for longer time (The longer the duration of purification, the greater the risk of protein degradation).
c) Add 1-10mM DTT prior to cell lysis (sometimes can increase significantly the binding); or try additives as glycerol, detergents or more NaCl
d) Purify protein under denaturating conditions.
e) Move tag to the opposite end of the protein.

If fusion protein is poorly eluted, try:
a) Decrease flow rate, or try overnight elution (The longer the duration of purification, the greater the risk of protein degradation)
b) Increase concentration of glutathione. First 15mM and then 20-40mM.
c) Increase ionic strength to 0.1-0.2M NaCl.
d) Increase the volume of elution buffer
e) Add a non-ionic detergent (as 0.1% Triton X-100 or 2% N-octyl glucoside) to reduce non-specific hydrophobic interactions that may prevent solubilization
    or elution of the fusion protein.

If multiple proteins bands are seen in the elution try:
a) If you suspect protein degradation (you can check previously with western blot using antibodies against GST or the fusion protein) try to work all the time at
    4°C and use protease inhibitors during lysis. (The longer the duration of purification, the greater the risk of protein degradation)
b) Decrease resin volume (allows higher competition between fusion protein and contaminants for the same sites on the resin)
c) Increase ionic strength to 0.1- 2M NaCl or use detergents that not destroy the protein activity during washing step
d) Increase the volume of the washing step.
e) Consider an additional purification step before or after purification.
f) A 70kDa protein (DnaK) sometimes co-purifies with the GST-fusion protein . To avoid co-purification: incubate before purification 10min 37ºC with  
    2mMATP 10mMMgSO4  and 50mMTrisHCl pH7.4 or remove after purification with ATP-Agarose or anion exchange

Protein degradation:

a) Discern if degradation happen or start during expression, or take place only during purification, or both of them.
b) Start during expression: can be seen by western-blot analysis of the protein integrity before purification (add sample buffer directlyon pellet cells). In this case,
    the options are: to change expression conditions, or to change bacteria strains or to modify amino acid sequence in the degradation site
c) Degradation start during purification: work quickly, 4C all the time, try to load on affinity column as soon as possible after lysis, since most of the proteases
    are in the crude lysate. If necessary, add more protease inhibitors (PI), or check which one avoid degradation, and strength only it (how to do it?: incubate in
    parallel a few hours crude lysate with or without different PI, and see which one is the better to avoid degradation. Once you know which one, you can
    strength your cocktail with this specific PI .
    Information about PI: ( http://wolfson.huji.ac.il/purification/extraction_and_clarification.html#Protease_Inhibitors) 
d) If you have a mix of problems and nothing helps, change construct for MBP, SUMO, etc

How to eliminate yellow color and endotoxins:
a) Yellow color in concentrated proteins can be eliminated with 100mM n-Octyl B-D-glucopyranoside (OGP):  incubate overnight and run a Gel Filtration
column. It can be used to remove yellow pigments and  endotoxins (LPS) too.
b) Endotoxins clearence:  wash  column with 50 column volumes of  buffer containing 0.1% (v/v) of TritonX-114 (Sigma-Aldrich, Germany) followed by
20 column volumes of  buffer without detergent at 4 °C  before elution {Timo Zimmerman et. al. Journal of Immunological Methods 314 (2006) 67–73}
c) Endotoxins can be removed with anion exchange columns, gel filtration, immobilized polymixin B, and other methods (see Endotoxin Removal)
For large scale production, the use of FPLC equipment with the proper resins will allow simple optimization and rapid and convenient comparison of protein
purification conditions.  
 

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