Introduction

      There is no reliable method for predicting the conditions needed to refold a protein. The folding of proteins in solution is affected by a number 
        of physiochemical parameters. These parameters include: Ionic strength, pH, temperature, oxidation state and protein concentration as well as 
        the presence of hydrophobic, polar, chaotropic agents and other proteins or factors. Thus, the identification of the conditions needed to properly 
        refold the protein remains completely empirical. For this purpose, many different Protein Refolding Screening Kits can be used:
        De Bernardez Clark et al. Methods in Enzymology 309: 217-236, (1999)
        Voziyan et al. J.of Pharmac.Science 89:1036-1045 (2000)
        Armstrong et al. Protein Science 8: 1475-1483 (1999)
        Willis et al. Protein Science 14: 1818-1826 (2005)
        Athenas-pdf , Novagen-pdf , Pierce-pdf , or any other commercial or published screen kits:

        Ionic Strength: from 50mM to 1M NaCl or KCl
        pH: from 4.0 to 9.0
        Temperature and Time
        Addtives:  1mM EDTA - 2mMCaCl₂ or 2mMMgCl₂  - 0.05% polyethylene glycol  3350 or 6000 - up to 0.75 M Guanidine HCl - up to1.5M Urea - 
            0.5 M arginine or Proline - L-Arg together with L-Glu at 50 mM - others     
        Sugars:  up to 1M Sucrose  - up to 0.5M Lactose -  up to 40% Glycerol  - up to 0.5M Trehalose - up to 1M TMAO - others.    
        Oxido-shuffling system: 1 mM GSH / 0.1 mM GSSH (or other concentrations) -  1-2mM TCEP, DTT  or BMC - others.                                                                    
        For proteins that needs to form disulfide bridges, a Redox system in the refolding buffer must be add.
        Detergents: ~0.5mM Tween 80 - ~0.12mM Tween 20 - up to1M NDSB - 0.3mM DDM - up to0.5% Triton X-100 - ~10mMCHAPS -
            Brij 35 up to 0.1% - others
        Detergents + ß-cyclodextrin (Machida et al. FEBS Letters 486: 131-135, 2000) (Oganesyan et. al. Journal of Structural and Functional Genomics 
            6: 177–182, 2005) (Veldkamp et al. Protein Expression and Purification 52: 202–209, 2007) 
        Other parameters

    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 refolding 
        and purification procedure and further scale-up.  Once you set up the best conditions at low scale, you can scale-up the procedure.

        Preparation of Inclusion Bodies (IB)

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.     Resuspend cell culture pellet with lysis buffer, (lysis in 1/10 or less, of original culture medium). 
        Suggested Lysis buffer : 20 to 50mM Na₂HPO₄ or TrisHCl or HEPES; pH 7.0 to 8.5
                                             300mM NaCl (could be from 0-1M NaCl)                          
                                             1mM PMSF + protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma) 
                                             Dnase 100U/ml or 25-50ug/ml (SIGMA DN-25). 
                                             Lysozime 0.2mg/ml.
                                             ßME, DTT or DTE  up to 10mM for proteins with many cysteines.
        Incubate 10min 4°C in the presence of 10mMMgCl₂.
        Gross filter to eliminate not resuspended particles

2.    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.

3.    Spin 20min 10000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet). 
       Keep sample of 40ul of supernatant for PAGE-SDS: soluble proteins

4.    Resuspend pellet in lysis buffer (use half of the volume of the first step) and keep sample of 40ul for PAGE-SDS: insoluble proteins, or 
       unlysed cells. Spin again.

5.    Repeat this last step at least once more.

6.    Detergent wash: resuspend pellet in lysis buffer (without DNaseI and lysozime) + 1% Triton X-100. Mix gently 5min. Spin.
        Repeat last step. If necessary wash with other detergents or denaturants. 
        See: Contaminant Removal from Inclusion Bodies Before Solubilization
        Repeated washes with denaturants or detergents can considerable reduce contaminant levels before IB solubilization.

7.     Repeat wash without detergent or denaturant

8.     Aliquot IB in many fractions. Keep at -70°C.  Check protein concentration.

        Solubilization and Refolding: Buffer Screening on Column

1.          Add solubilization buffer (50 mM Tris-HCl pH 8.75 + 6 M GdmCl.) to the inclusion body pellet to give a protein concentration of 2 mg/mL.
For proteins that needs to form disulfide bridges, add Beta Mercapto-ethanol from the 14 M stock up to 15 mM.
Mix thoroughly and leave at room temperature for 60 min to ensure complete solubilization and reduction of the recombinant protein.

2.         Remove any non-solubilized material by centrifugation at 12500 RPM for 5 min at 4°C.

3.     Mix supernatant of last step, with equilibrated Ni resin (equilibrated with solubilization buffer) by gentle agitation 1hr at 4°C
Use 1ml resin (2ml suspension) for each 10mg protein

4.          Spin 5’ 4000rpm 4◦C. Separate unbound from resin

5.          Wash resin three times during 2 min each by 10 volumes of Ni wash buffer (containing 8 M urea) + 10 mM imidazole and 12 mM Beta-mercaptoethanol.
Check PAGE-SDS of  samples of each step before elution to guarantee that the protein was not lost before the elution.

        Imidazole concentrations can be increase in the washing buffer (to 20, 30, 40mM, or more) to eliminate weak Ni binding contaminats proteins. 
Check this last point by performing a parallel protein refolding experiment using differents Imidazole concentration in the washing step. 
Ni binding contaminats proteins can be eliminated too by increasing  volume and number of washing steps.

6.          Ten times excess of the refolding buffer was added to the resin, the protein was allowed to refold during 1 h at constant agitation at 4°C, and then leave    
        ON 4°C.   According to a modification of O.A. Sharapova, et al., J. Chromatogr. A (2011), doi:10.1016/j.chroma.2011.05.075     

7.          Nex t day spin 5’ 4000rpm 4°C. Separate unbound from resin.

8.          Elute two times with two volumes of 50mM Tris + 500 mM NaCl + 300 mM imidazole pH 7.5 (incubate 3 to 5min each time before spinning at 4°C).

9.    Keep samples ON 4°C. Check turbidity of the samples. Spin 12500 RPM for 20 min at 4°C.  Check PAGE-SDS of  supernatant. 

10.  Check oligomeric state (aggregation) of the relevant samples by analytical GF (without turbidity and more protein according to the last PAGE-SDS.

11.  Optimize in next experiments: type and quantitie of  additives, sugars, detergents, etc. Their use during elution. Final storage buffer.

    Solubilization and Refolding: on-Column Scale-up. Automatization

1.          Add solubilization buffer (50 mM Tris-HCl pH 8.75 + 6 M GdmCl.) to the inclusion body pellet to give a protein concentration of 2 mg/mL.
Mix thoroughly and leave at room temperature for 60 min to ensure complete solubilization and reduction of the recombinant protein.

2.         Remove any non-solubilized material by centrifugation at 12500 RPM for 5 min at 4°C.

3.     Mix supernatant of last step, with equilibrated Ni resin (equilibrated with urea buffer: 50 mM Tris-HCl pH 7.5 + 500 mM NaCl + 6 M Urea ) 
by gentle agitation 1.5hr at 4°C. Batch binding could be better that direct loading on column, to avoid high protein concentration on top of the column.
Use 1ml resin (2ml suspension) for each 10mg protein

4.          Spin 5’ 4000rpm 4◦C. Separate unbound from resin.