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 Amylose resin

Place 200µl beads (400µl suspension) of Amylose resin in 1.5ml plastic tube. According to New England BioLabs the resin binds 3mg 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

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) 10mM ß-ME, or 1mM DTT or 1mM DTE  for proteins with many cysteines.
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) 0.02% NaN₃ (azide)

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

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

Regeneration of Amylose resin

According to New England BioLabs the resin 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.

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 Amylose resin.

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.

If protein does not bind to the Amylose resin, there are several options to choose:
a) Check the Amylose 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 amylase in the crude material can affect the performance of the resin; try to use glucose in the media (repressed the amylase)
e) Construct a new vector with the tag in 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 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

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 MBP 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 up to 1M NaCl or KCl.
d) Increase the volume of the washing step.
e) 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.  
 

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