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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@mail.ls.huji.ac.il  Tel: 972-2-6586920 

Small scale His-Tag fusion protein purification under denaturative conditions


Introduction

High levels of expression of recombinant proteins in a bacterial system can lead to the formation of insoluble aggregates, usually known as inclusion bodies (IB). 6M Guanidine-HCl (GuHCl), 8M Urea or other strong denaturants can be used to completely solubilized IB. Since under denaturating conditions the His tag is completely exposed, it will facilitate the binding to Ni columns. For most biochemical studies, protein have to be renatured and refolded, and this can be done usually after elution or sometimes in the column itself before elution.
We describe here two protein purification procedures. Procedure-I, where the protein is extracted from the bacterial pellet in the presence of denaturating agents and purified on Ni2+  beads; or Procedure-II where the protein is solubilized from partially clean or clean IB (see Contaminant Removal from Inclusion Bodies Before Solubilization) before purification on Ni2+ beads. The second procedure although more laborious, can help to reduce the background of  protein contaminants in the final purification step.
  

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 equilibration buffer (washing: mix, spin 3min 3500rpm, discharge supernatant).
 
 

Buffers

Lysis buffer: 50mM Na2HPO4 pH 8.0, 0.3M NaCl, 1mM PMSF (or protease inhibitor cocktail for bacterial cells #P-8849 from Sigma) and strong denaturant as  6M Guanidine-HCl (GuHCl) or 6 to 8M Urea
    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) ßME up to 20mM
as a reducing agent if the protein has Cysteines.

Equilibration buffer: 6 to 8M Urea, 50mM Na2HPO4 pH 8.0, 0.5M NaCl

Washing buffer: 6 to 8M Urea, 50mM Na2HPO4 pH 8.0, 0.5M NaCl

Elution buffer:  6 to 8M Urea, 20mM Tris pH 7.5, 100mM NaCl, and appropriate imidazole concentrations.

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

Procedure I: Protein Extraction from Bacterial Pellet in the presence of a strong denaturant

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

2   Prepare lysis buffer containing urea 6 to 8M or Guanidine-HCl  6M (try 8M of Urea first, and if protein is soluble, titer down in the next experiments till minimal urea concentration is required for protein solubilization)

3   Sonicate on ice 3 x 20 seconds (depends of the sonicator)

4   Spin 15min max speed 4°C

5   Transfer supernatant into clean tube: crude extract (keep 40µl for PAGE-SDS)

6   Equilibrate 50µl Nil beads with equilibration buffer (see Equilibration of Ni-NTA agarose)

7   Add the crude extract to the beads and incubate 4°C / 1h (swirl)

8   Spin 3min 3500rpm. Dicharge unbound material (keep 40µl for PAGE-SDS)

9   Wash 3x1ml with wash buffer with appropriate urea concentration. Washing: mix, spin 3min 3500rpm, discharge supernatant (keep 40µl for PAGE-SDS)

10  Wash 2x1ml with wash buffer + 10mM imidazole (keep 40µl for PAGE-SDS)

11  Elute with 3x100µl elution buffer + 250mM imidazole (keep 40µl for PAGE-SDS) (elution: mix, keep 3min at 4°C, spin 3min 3500rpm, collect supernatant)

12  Run on PAGE-SDS gel 5µl of crude extract and unbound material, and 13µl of the wash and elution fractions. 


Procedure II: Protein Extraction from Inclusion Bodies (IB) in the presence of a strong denaturant

1   Resuspend pellet of 10ml bacterial culture (or 100ml bacterial culture for very low expression level) in 1ml lysis buffer without denaturants as Urea or Guanidine-HCl

2   Sonicate on ice 3 x 20 seconds (depends of the sonicator) 

4   Spin 15min max speed 4°C. Separate supernatant before solubilization (keep 40µl for PAGE-SDS)  from pellet (IB).

5   Resuspend IB in 0.5ml lysis buffer containing urea 6 to 8M or Guanidine-HCl  6M (keep 30min at 30°C) or wash IB before solubilization as suggested in ""Contaminant Removal from Inclusion Bodies Before Solubilization"" (this step although more laborious, can help to reduce the background of  protein contaminants in the final purification step). After IB solubilization spin 15min max speed 4°C. Separate supernatant after solubilization (keep 40µl for PAGE-SDS)  from insolubilized pellet (suspend pellet in 0.5ml lysis buffer and keep 40µl for PAGE-SDS). Transfer supernatant into clean tube

6   Equilibrate 50µl Nil beads with equilibration buffer (see Equilibration of Ni-NTA agarose)

7   Add the last supernatant extract to the beads and incubate 4°C / 1h (swirl)

8   Spin 3min 3500rpm. Dicharge unbound material (keep 40µl for PAGE-SDS)

9   Wash 3x1ml with wash buffer with appropriate urea concentration. Washing: mix, spin 3min 3500rpm, discharge supernatant (keep 40µl for PAGE-SDS)

10  Wash 2x1ml with wash buffer + 10mM imidazole (keep 40µl for PAGE-SDS)

11  Elute with 3x100µl elution buffer + 250mM imidazole (keep 40µl for PAGE-SDS) (elution: mix, keep 3min at 4°C, spin 3min 3500rpm, collect supernatant)

12  Run on PAGE-SDS gel 5µl of supernatant extract before and after solubilization, insolubilized pellet, and unbound material, and 13µl of the wash and elution fractions. 


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+
 

Analysis of results - Troubleshooting

If protein does not bind to the Ni-NTA resin, check the integrity of the His tag  by western blot using anti-polyhistidine antibodies, or do N-terminal sequencing in the case of N-terminal tags. Try to work at 4°C all the time using protease inhibitors during lysis.

If target protein elute with protein contaminants Try to load solubilized clean IB as indicated in the Procedure-II. Or try the alternative protocol with increasing Imidazol concentration. Or reduce amount of Ni-NTA resin. Or try additives as ßME, glycerol, detergents or more NaCl in the washing / elution buffers.
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 eluted protein seem to be degraded try to work at 4°C all the time and use protease inhibitors during lysis.

If the protein does not elute from the column (protein is attached to the column) use higher Imidazol concentrations (up to 1M).
 
 
 

Alternative protocol if target protein is not pure enough

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 Imidazol.

Check eluted proteins on PAGE-SDS. Expect lower yields but higher purification by increasing the Imidazol concentration
 
 
 
 
 
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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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