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

Small and large 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 IMAC resin

Place 50ul beads (100ul suspension) of Ni-NTA agarose beads  (or any other commercial 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 low Scale

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

Suggested buffer: 25mM TrisHCl/NaPO4 pH 8.0  + 0.3M NaCl +  "optional" additives
Alternative buffers: 20-50mM TrisHCl, MOPS, HEPES or Phosphate  buffers from pH 7.0 to 8.0 and NaCl or KCl  
from 25mM to 1M
                                  
Optional additives to the lysis buffer

a) ßME up to 15mM for proteins with cysteines residues. Maintain reduced cysteines and avoid the formation of
nonspecific disulfide bridges that can cause aggregation (not recommended if you want to mantain disulfide bridges).
b) 0.1-2% Triton X-100, NP40, or any other detergent that do not affect the biological activity of your protein.
c) 0.02% NaN3 (azide) to avoid bacterial contamination in the buffer
d) Up to 5-40mM Imidazole to reduce non-specific binding to the column. Initial Imidazole concentration can be
check previously at low scale: see alternative protocol 
e) Glycerol 10% to stabilize protein and avoid aggregation 

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

Recommended additives to the lysis buffer:

f) Dnase 100U/ml or 25-50ug/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of 10mMMgCl2
g) Lysozime 0.2mg/ml. Incubate 10min 4°C
h) 1mM PMSF and/or protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma or any
other commercial cocktail without EDTA)

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 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, and extensive sonication).

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 buffer and keep sample of 40ul for PAGE-SDS: insoluble proteins, or unlysed cells.
 
 

Protein Purification low Scale 

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

6) Spin 3min 3500rpm 4°C. Discharge supernatant and keep sample of 40µl for PAGE-SDS: unbound proteins

7) Wash beads with 1ml buffer several times (washing: mix, spin 3min 3500rpm, keep supernatant aside, be careful not to
take the resin) up to OD280nm <0.05. Keep sample of 40µl for PAGE-SDS of each washing.

8) Elute recombinant protein with 3x100ul buffer + 250mM Imidazol  (incubate 3 to 5min each time before spinning 3min,
3500rpm at 4°C). Keep sample of 40ul for PAGE-SDS of each elution.

9) 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).

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

11)  Optimize purification according to results before large scale purification (see Troubleshootings and Alternative protocol)


Large Scale Extraction and 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 GE-Healthcare  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:
A) gradient or step gradients elutions
B) optimization of flow rate, column dimension, washing conditions, etc.
C) 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+
You can consider to use IMAC as a first capture step, or as an intermediate step after a capture step with ion or hydrophobic
exchange chromatography

VERY IMPORTANT

A.   MANTAIN PROTEIN AT 4ºC DURING ALL THE PROCEDURE
B.   AVOID  PROTEIN DENATURATION BY FROATHING (FOAM)
C.    WORK AS QUICKLY AS YOU CAN TO AVOID PROTEOLYTIC DIGESTIONS
D.    Keep sample for PAGE-SDS from each step

1.     Equilibrate IMAC column with buffer (mg fusion protein per ml bed volume depends of the commercial resin). Equilibration is
confirmed by measuring pH and conductivity. Highest flow-rate and pressure limit: depends of the commercial resin

2.     Resuspend cell culture pellet with suggested lysis buffer, (lysis in 1/10 or less, of original culture medium). Initial Imidazole
concentration during binding and washing  steps, can be check previously at low scale: see alternative protocol
        Incubate 10min 4°C in the presence of 10mMMgCl2.
        Gross filter to eliminate not resuspended particles

3.    Microfluidizer or French Press lysis at 21000psi (less recommended: Sonication 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.

4.    Spin 20min 10000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet).
Filter supernatant with GF/D (Whatman) and 0.45 mm filter (Whatman). Keep sample of 40µl of supernatant for PAGE-SDS:
soluble proteins, and from insoluble proteins, or unlysed cells (use resuspended pellet)

 5.    Load supernatant on equilibrate IMAC column at low flow rate (Suggestion: loading time ~1.5hr); wash with buffer at higher
flow rate up to low 280nm optical density. Keep sample of 40µl of supernatant for PAGE-SDS: unbound and wash fractions.
Imidazole concentration during washing steps, can be check previously at low scale: see alternative protocol

6.     Protein is eluted by step-gradient, using as elution buffer: buffer + 300mM Imidazole:
        a) Low concentration Imidazole steps at lower flow-rate (start collecting fractions) to eliminate low binding contaminant proteins.
        Suggested steps: 10, 20 and 30mM Imidazole in buffer.
        b) Short gradient up to 300mM Imidazole at very low flow-rate in order to obtain sharp peak, up to low 280nm optical density.
        Collect smaller fractions.
        c) Samples from each step and each fraction are analyzed for protein content by SDS–PAGE. Protein-containing fractions are
        then pooled according to the profile obtained.
        d) Optimize next purification and scale-up, according to results (optimization by adding, changing or eliminating steps)

7.     Protein is 70–90% pure following this single-capture step. In order to achieve a higher degree of purification, which is often
required for downstream applications such as structural studies, one should add additional purification steps such as ion exchange,
hydrophobic exchange, and size exclusion chromatography. Ion exchange chromatography is essential as an intermediate step for
separating target proteins from protein contaminants such as chaperons  and other host cell proteins. It also allows one to separate
the target protein from heterogeneously folded forms that are a consequence of  the expression and purification conditions used and
from heterogeneity in posttranslational modifications. Sometimes purification techniques that separate proteins according to their
charge are insufficient, and other approaches based on different principles, such as hydrophobic exchange chromatography or
hydroxyapatite, should be used. As a final polishing step, it is often recommended to use size-exclusion chromatography, not only
to eliminate protein contaminants and low molecular weight molecules but also to obtain a homogeneous oligomeric form.
An added value of the gel filtration step is that the protein will elute in the final desired buffer.

8.     When His-Tag fusion proteins are purified by affinity chromatography without further purification columns, dialysis after affinity
purification is enough to eliminate Imidazole from the protein solution. Other possibility to eliminate Imidazole: binding the fusion protein
to hydroxyapatite, by ion exchange, hydrophobic exchange, or any other resin that can bind the fusion protein and not Imidazole.
The resin is then washed extensively before protein elution. Similar approach can be done for buffer exchange or detergent exchange.

Regeneration of IMAC 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 IMAC resin.

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 IMAC 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, different detergents up to 2% or increase ionic strength up to 1.5M NaCl or KCl in the lysis buffer.
Try low concentration of denaturants like Urea up to 1.5M or Guanidine HCl up to 1M
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 IMAC column through Ion or
Hydrophobic Exchange Chromatography.

If multiple proteins bands are seen in the elution try:

a) If multiple protein bands are present after elution, then protein degradation is to be suspected. Western blot analysis can be
performed to verify if proteolysis is occurring. Conducting all purification steps at 4°C, reducing the overall time taken to carry
out the procedure, and using protease inhibitors during the cell disruption process, can all help to reduce proteolysis.
b)  If the additional bands visible on SDS–PAGE are not the result of target protein degradation, there are two main reasons that
usually explain the presence of cellular protein contaminants: (i) contaminating proteins are binding nonspecifically to the resin,
(ii) contaminants are sticking to the target protein. If contaminants are bound nonspecifically to the resin, consider decreasing the
resin volume to increase competition, or increasing the ionic strength of the buffers (up to 1 M NaCl or KCl), to reduce hydrophobic
interactions with the resin; or use more stringent conditions during binding and washing by increasing Imidazole concentrations:
see alternative protocol
c) If contaminants stick to the target protein, increasing the washing step is the first option that should be considered. If this does not
work, consider  increasing the ionic strength of the buffers (up to 1 M NaCl or KCl), adding additives such as glycerol, adding up to
15mM ß-ME as reducing  agents in order to disrupt nonspecific intermolecular disulfide bonds, or adding detergents as Triton X-100,
NP40 or Tween 20 up to 2% that  might reduce hydrophobic interactions. 
d) If taking these options does not reduce the presence of contaminants, additional purification steps should be performed before or
after affinity purification. In some cases, columns charged with other metal ions instead of  Ni2+ (Zn2+, Co2+, Fe2+ and Cu2+) can give
a different purification pattern, or IMAC resins of different suppliers.
e) 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.
f)  Robichon C. et al ( APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2011, p. 4634–4646  Vol. 77, No. 13)
engineer Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by Immobilized
Metal Affinity Chromatograph [two new strains, NiCo21(DE3) and NiCo22(DE3)], which express the endogenous Ni contaminants
proteins fused at their C terminus to a chitin binding domain (CBD), so then can be eliminated by chitin resins; and replace with alanines
six surface histidines of another contaminant protein. They claim a significant reduction of Ni impurities using these strains  (pdf)

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 Ni column with 50 column volumes of  buffer containing 0.1% (v/v) of TritonX-114 (Sigma-Aldrich)
followed by 20 column volumes of  buffer without detergent at 4 °C   {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)

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 (see supplier recommendations)
NaCl or KCl up to 2M.
 

Alternative protocol for low scale purification if target protein is not pure
enough (example)

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

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

3) Check eluted proteins on PAGE-SDS. Expect lower yields but higher purification by increasing the Imidazole concentration.
 See example. 

4)  Results of this protocol can be use to choose the Imidazole concentration to be use during binding and step washings in large scale
purification using IMAC columns
 
 
 
 
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Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mariol@cc.huji.ac.il  Tel: 972-2-6586920  

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