The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker
mariol@mail.ls.huji.ac.il  Tel: 972-2-6586920 

Small scale His-Tag fusion protein purification under nature conditions

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.
 

Example:
1)    Grow 1L culture
2)    Induce (IPTG, salt induction, etc. etc.)
3)    Spin cell culture 10min 8000rpm 4ºC, discharge supernatant
4)    Resuspend cell pellet at 4ºC very gently with 100ml cold PBS buffer. Aliquot as following:
            a) 10 tubes (1.5ml plastic tubes) with 1ml suspension (it means 10ml original culture per tube);
            b) 4 tubes (15ml plastic tubes) with 10ml suspension (it means 100ml original culture per tube)
            c) 1 tube (50ml plastic tube) with 50ml suspension (it means 500ml original culture).
5)    Spin 10min 8000rpm 4ºC, discharge supernatant
6)    Keep cell pellet at -80ºC


Equilibration of Ni-NTA agarose

Place 50ul beads (100ul suspension) of Ni-NTA agarose beads 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

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

Suggested Lysis buffer: 50mM TrisHCl/NaPO4 pH 8.0 (could be TrisHCl alone or NaPO4 pH 7.5-8.0) + 0.3M NaCl (optional 0.1-0.5) +  "optional" additives
                                  
Optional additives to the lysis buffer

a) Protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma) or 1mM PMSF

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 up to 15mM to reduce free Cysteines (not recommended if you want to mantain disulfide bridges).

e) 0.1-2% Triton X-100, NP40, or any other detergent that do not affect the biological activity of your protein.

f) 0.02% NaN3 (azide)

g) Up to 20-40mM Imidazole to reduce non-specific binding to the column (sometimes can affect binding) 

Reagents compatible with the purification:
 
6M Guanidine HCl 2% Tween 20 50% Glycerol 4M MgCl2
8M Urea 1% CHAPS 20% Ethanol 5mM CaCl2
2% Triton X-100 20mM ßME 2M NaCl at 20mM Imidazole

Limitations

Do not exposed Ni matrices to reducing agents as DTT or DTE ( you can use up to ßME 20mM); chelating agents as EDTA and EGTA; NH4+ buffers and amino acids as Arg, Glu, Gly or His.

2) Sonicate in ice bucket 3 x 10 sec (or more if the cells are not completely disrupted)

3) Spin 10min 13000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet). Use supernatant for next step. Keep a 40ul sample of supernatant for PAGE-SDS:soluble proteins

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

Protein Purification

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

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

3) Wash beads with 2 x 1.5ml washing buffer : 50mM NaPO4 / TrisHCl pH 8.0; 0.3M NaCl ("optional": up to 20mM Imidazole). Keep sample of 40ul for PAGE-SDS of each wash.

4) Elute recombinant protein with 3x100ul wash buffer + 250mM Imidazol  (incubate 3 to 5min each time before spinning 3min, 3500rpm at 4°C). Elution buffer: as washing buffer + 250mM Imidazole.

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: proteinnot eluted (or SDS extracted beads).

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


Large Scale

The procedures described here are for low scale purification. If larger amounts of proteins are to be purified we recommend the use of open columns or FPLC equipment with resins that can be used at high pressure: like Ni-NTA superflow resin from QIAGEN or BD TalonTM Metal Affinity from CLONTECH or Chelating Sepharose Fast Flow or Ni Sepharose High Performance from AMERSHAM-BIOSCIENCES or Ni-NTA Hi-Bind or Metal Chelate Resins from NOVAGEN-MERCK .
The use of FPLC equipment will allow greater operational flexibility and simple optimization:
1) gradient or step gradients elutions
2) optimization of flow rate, column dimension, washing conditions, etc.
3) rapid and convenient comparison of protein purification by the use of  columns charged with other metal ions with different strength of binding, e.g. Zn2+ , Co2+, Fe2+ and Cu2+


Regeneration of Ni-NTA resin

According to QIAGEN the resin may be reused many times when regenerated promptly after use, and should be performed with identical recombinant proteins.

Short wash after each run
1) Highly suggested: NaOH 0.5M 5cv (column volumes) and buffer up to neutral pH
2) Water 5cv
2)  If storage for more than 1-2 days: 20% Ethanol wash with 3cv and keep at 4°C.
     If storage for less than 1-2 days: Wash or lysis buffer + 0.02%NaAzide, wash with 3cv and keep at 4°C.

Short regeneration after several runs
1) Water 5cv (column volumes)
2) EDTA 100mM pH8.0  5cv
3) Water 5cv
4) NiSO4 100mM  2cv (Recharging)
5) Water 5cv 
6)  If storage for more than 1-2 days: 20% Ethanol wash with 3cv and keep at 4°C.
     If storage for less than 1-2 days: Wash or lysis buffer + 0.02%NaAzide, wash with 3cv and keep at 4°C.

More stringent regeneration for a highly contaminated column (According to QIAGEN)
1)  Regeneration buffer (6M GuHCl or 0.2M Acetic acid) 2cv (column volumes)
2)  Water 5cv
3)  2%SDS 3cv
4)  25% Ethanol 1cv
5)  50% Ethanol 1cv
6)  75% Ethanol 1cv
7)  100% Ethanol 5cv
8)  75% Ethanol 1cv
9)  50% Ethanol 1cv
10) 25% Ethanol 1cv
11) Water 5cv (column volumes)
12)  EDTA 100mM pH8.0  5cv
13) Water 5cv
14) NiSO4 100mM  2cv (Recharging)
15) Water 5cv 
16)  If storage for more than 1-2 days: 20% Ethanol wash with 3cv and keep at 4°C.
     If storage for less than 1-2 days: Wash or lysis buffer + 0.02%NaAzide, wash with 3cv and keep at 4°C.

 
 

Analysis of results - Troubleshooting

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

If most of the protein remains insoluble after extraction, try
a) To change lysis buffer by adding additives as ßME, glycerol, detergents or more NaCl.
b) Re-extract pellet with more buffer,
c) Use more lysis buffer during extraction,
d) Perform a more intensive sonication,
e) Incubate with lysozyme before sonication.
f) Try the denaturating protocol

If protein does not bind to the Ni-NTA resin, there are several options to choose:
a) Check the NI-NTA resin: binding of a cell sonicate containing a control protein
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) Try additives as glycerol, detergents or more NaCl
d) His-tag is inaccessible, purify protein under denaturating protocol
e) Check by western-blot if the His-tag has been degraded; if this is the case, try to work all the time at 4°C and use more protease inhibitors during lysis
f) Construct a new vector with the tag in the opposite end of the protein. 
g) According to (A.Magnusdottir et al.) a serious drawback of IMAC is the often-experienced failure to purify low-abundance His-tagged proteins from E. coli lysates; 
    increasing the culture size and thereby increasing the amount of available His-tagged protein does not result in increased yield. They examined this issue and propose 
    that it is tightly linked to metal-ion leakage from the columns induced by periplasmic material from the E.coli lysate, and this periplasmic fraction can be removed by 
    osmotic shock (A.Magnusdottir et al.B) .
    Another possibility is to perform a purification step before the Ni column through Ion or Hydrophobic Exchange Chromatography.

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 His-tag 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) Use more stringent conditions during binding and washing:  try the alternative protocol with increasing Imidazole concentrations.
c) Decrease resin volume (allows higher competition between fusion protein and contaminants for the same sites on the resin)
d) If contaminants are associated with the tagged protein, try to disrupt the non-specific interaction by adding to the wash buffer before elution  additives as ß-ME, glycerol up to 50%, detergents as Triton X-100, NP40 or Tween 20 up to 2% or increase ionic strength up to 1.5M NaCl or KCl.
e) Increase the volume of the washing step.
f) Consider an additional purification step before or after purification.
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. 

If the protein does not elute from the column
a) Use higher Imidazole concentrations (up to 1M), or additives
b) Reduce elution flow-rate
c) Elute under denaturating conditions


Additives

ßME up to15mM.

Glycerol up to 50%.

Detergents that not affect the protein activity.

NaCl or KCl up to 2M.
 
 

Alternative protocol if target protein is not pure enough (example)

Perform parallel purification procedures where you include 10-20-30-40 or 50mM Imidazole in the lysis,binding and washing buffer.

Elute directly with 3x100ul elution buffer + 250mM Imidazole.

Check eluted proteins on PAGE-SDS. Expect lower yields but higher purification by increasing the Imidazole concentration. See example.
 
 
 
 
 
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Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mariol@mail.ls.huji.ac.il  Tel: 972-2-6586920  

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