Small scale His-Tag purification under nature conditions

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 MBP-fusion Protein Purification
See 1) New England BioLabs Instruction Manual (pdf-I) (pdf-II)
2) GE-Healthcare Instruction Manual (pdf-I) (pdf-II) (pdf-III)

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 Amylose or Dextrin Sepharose resin

Place 200µl beads (400µl suspension) of Amylose resin in 1.5ml plastic tube (According to New England BioLabs the resin binds
3 mg fusion protein per ml bed volume) or Dextrin Sepharose™ High Performance (According to GE-Helthcare binds 7-16 mg
fusion protein per ml bed volume)

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

Protein Extraction low scale

1) Resuspend pellet of 40ml cell culture in 5ml lysis buffer.

Suggested Lysis buffer : 200mM NaCl; 20mM TrisHCl pH 7.4; 1mM EDTA (COLUMN BUFFER) and additives
Alternative buffers: MOPS, HEPES and Phosphate buffers around pH 7.0 and NaCl or KCl from 25mM to 1M.

Optional additives to the lysis buffer
a) 1mM PMSF and/or protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma or any other
commercial cocktail)
b) Dnase 100U/ml or 25-50µg/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of 10mMMgCl₂.
c) Lysozime 0.2mg/ml. Incubate 10min 4°C
d) 0.02% NaN₃ (azide) to avoid bacterial contamination in the medium
f) 5mM ß-ME, or 1mM DTT 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)
e) According to New England BioLabs the use of non-ionic detergents as Triton X-100, NP40 or Tween-20 "could"
interfere with the binding of the
fusion protein to the affinity resin
f) Glycerol 10% to stabilize protein and avoid aggregation

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).

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

4) Resuspend pellet in 5ml column buffer and keep sample of 40µl for PAGE-SDS: insoluble proteins, or unlysed cells.

Protein Purification low scale

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 40µl for PAGE-SDS: unbound proteins (this material
could be use again in case of overloading).

3) Wash beads with 5ml column buffer several times (washing: mix, spin 3min 3500rpm, keep supernatant aside, be careful not to
take the resin) up to OD280nm <0.05 of column buffer. Keep sample of 40µl for PAGE-SDS of each washing.

4) Elute recombinant protein with 300µl elution buffer (column buffer + 10mM Maltose) several times up to OD280nm <0.05 of
elution buffer (mix gently, incubate at RT for 5min for each elution, spin 3min 3500rpm , keep supernatant aside).
Keep sample of 40µl for PAGE-SDS of each elution.

5) Resuspend 5µl beads in 10µl H₂O + 3µl 5x sample buffer. Mix and spin. Keep sample of 40µl 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
MBP alone: 42100

Large Scale Extraction and Purification

If larger amounts of proteins are to be purified we recommend the use FPLC equipment with resins like Dextrin Sepharose™
High Performance from GE. The use of FPLC equipment will allow greater operational flexibility and simple optimization:
optimization of flow rate, column dimension, washing conditions, etc. You can consider to use Dextrin Sepharose 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 Dextrin Sepharose™ High Performance column with buffer (consider ~5 mg fusion protein per ml bed volume).
Equilibration is confirmed by measuring pH and conductivity. Pressure limit: 0.5 MPa

2.   Resuspend cell culture pellet with suggested lysis buffer, (lysis in 1/10 or less, of original culture medium).
        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 Dextrin Sepharose™ HP column at low flow rate (at least 1.5hr); wash with buffer at higher
flow rate up to low optical density. Keep sample of 40µl of supernatant for PAGE-SDS: unbound and wash fractions.

6.     Protein is eluted with elution buffer (buffer + 10 mM maltose) at low flow rate, to get sharp peak. 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.

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 MBP fusion proteins are purified by affinity chromatography without further purification columns, dialysis after affinity
purification is not enough to eliminate maltose from the protein solution. Maltose can be completely removed by binding the fusion
protein to hydroxyapatite, or by ion exchange, or hydrophobic exchange, or any other resin that can bind the fusion protein and not the
sugar. The resin is then washed extensively before protein elution (see pMAL™ Protein Fusion and Purification System manual from
NEB)

Regeneration of Amylose or Dextrin Sepharose resin

Amylose resin from New England BioLabs may be reused 3-5 times when regenerated promptly after use.
1) Water 3cv (column volumes)
2) 0.1%SDS 3cv
3) Water 5cv
4) If storage for more than 1-2 days: wash with 3cv and keep with 20% Ethanol.
If storage for less than 1-2 days: wash with 3cv and keep with column buffer + 0.02%NaAzide.

Dextrin Sepharose™ High Performance may be reused many times when regenerated promptly after use.
1) Water 3cv (column volumes)
2) 0.5M NaOH several cv or 0.1%SDS 3cv
3) Neutralize with buffer and then water 5cv for NaOH or water 5cv for SDS
4) If storage for more than 1-2 days: wash with 3cv and keep with 20% Ethanol.
If storage for less than 1-2 days: wash with 3cv and keep with column buffer + 0.02%NaAzide.

Further Purification of the Protein target from MBP after Factor Xa cleavage

Factor Xa cleavage of the protein target from MBP:
The MBP fusion protein can be cleavage with factor Xa (see protocol: Factor Xa Cleavage of MBP-Fusion protein). Factor Xa
cleavage site: Ile-Glu-Gly-Arg; but there are many examples where Factor Xa can cleavage in other sites.
Cleavage can be performed using the free intact fusion, or in same cases with the fusion protein bound to a matrix.
The amount of factor Xa, temperature and length of incubation must be calibrated for each system. Samples must be removed at various
time points and analyzed by PAGE-SDS to estimate the yield, purity and extent of factor Xa digestion.
Theoretically, cleavage must be complete following ON treatment at RT with 10-30 µg Factor Xa from New England
BioLabs per mg fusion protein.
For some applications factor Xa (MW 43kDa consisting of two subunits of 27 and 16kDa) must be subsequently removed from the
sample by chromatography or by shaking or rotating at 22°C (or RT) 30 min with pAminoBenzamidine - Agarose (SIGMA #A 7155)
(AmershamBiosciences #17-5123-10).
Factor Xa inactivation: 2µM dansyl-Glu-Gly-Arg-chloromethyl ketone (CALBIOCHEM #251700) or1mM PMSF.

How to separate the protein target from MBP after cleavage:
MBP MW is 42kDa and the theoretical pI: 5.07
1) If the MW of the target protein and MBP are considerable different, you can use gel filtration chromatography to separate both of them.
2) Dialysis to eliminate maltose, and repurification through Amylose resin.
3) Anion exchange chromatography (like DEAE or Q-Sepharose FF from Amersham Bioscience) using an NaCl gradient from 25mM
to 1M in 20mM TrisHCl pH8.0 According to New England BioLabs MBP will elute at 100-150mM NaCl and Factor Xa at
around 400mM NaCl.

Analysis of results - Troubleshooting

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

If most of the protein remains insoluble after extraction:
a) Add ßME, DTT, glycerol, detergents or more NaCl
b) Re-extract pellet with more buffer
c) Use more lysis buffer during extraction
d) Perform a more intensive cell disruption
e) Incubate with lysozyme before cell disruption

If protein does not bind to the Affinity resin, there are several options to choose:
a) Check the resin: binding of a cell sonicate containing only MBP
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, or more NaCl
d) The presence of nonionic detergents such as Triton X-100 and Tween-20 can interfere with binding. If detergents are essential to the
    target protein, use less than 0.05% in order to solubilize the extract. However, if this concentration is too low, you might need to consider
    improving binding by screening alternative detergents.
e) The oligomeric state of the molecule (soluble aggregates) can affect its binding to affinity columns. The presence of soluble aggregates
    can be analyzed by gel filtration. The formation of oligomers can be reduced by changing the expression conditions or the purification
    procedure and by screening different buffers and additives.
f) Consider adding a polyhistidine tag (His6) to the N terminus of MBP. This addition does not interfere with the ability of MBP to promote
    the solubility and proper  folding of its fusion partners, and it can be used for binding to the more commonly used immobilized metal affinity
    chromatography systems (IMAC)
g) The presence of amylase in the crude material can affect the performance of the resin; try to use 0.2% glucose in the media
    (repressed the amylase)
h) Construct a new vector with the tag in the opposite end of the protein.

If fusion protein is not eluted efficiently:
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 maltose in the elution buffer. First 20mM and then 50-100mM.
c) Increase ionic strength up to 1M NaCl or KCl.
d) Increase the volume of elution buffer
e) The oligomeric state of the protein can change as a result of the high protein concentration in the column. Here, changes in the buffer can
    prevent aggregation and following options should be considered: (i) increasing ionic strength up to 1 M NaCl or KCl, (ii) adding detergents
    or additives such as glycerol to the buffers, and (iii) performing batch binding instead of column binding.

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.
    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 reducing agents
    in order to disrupt nonspecific intermolecular disulfide bonds, or adding detergents that might reduce hydrophobic interactions.
c) If taking these options does not reduce the presence of contaminants, additional purification steps should be performed before or after
    affinity purification.

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, endotoxins and NADH oxidation contamination activity :

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)
d) Contaminating NADH oxidation activity could be eliminated through the addition of 0.1% Triton X-100 and 2% glycerol to the column
    buffer during homogenization of bacteria and first column wash, followed by an additional wash and elution with regular column and elution buffers.
    Or by IMAC columns if the fusion protein contains a His-tag (Biotechniques. 52(4): 247-53 (2012) Guo F, Zhu G.)

HOME EXPRESSION SYSTEMS EXTRACTION AND CLARIFICATION PURIFICATION CHARACTERIZATION OTHERS

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