Cleavage of GST Fusion Proteins With PreScission Protease
Protocol09/08/2020 6:09 pm

Cleavage of GST Fusion Proteins With PreScission Protease

 

 

 

Introduction: 

 

Recombinant proteins are commonly expressed as translational fusions to Glutathione S-transferase (GST). GST is a naturally occurring 26 kDa protein found in eukaryotic cells. The GST moiety binds with high affinity to glutathione coupled to a Sepharose matrix (Glutathione Sepharose). This binding is reversible and the protein can be eluted under mild, non-denaturing conditions by the addition of reduced glutathione to the elution buffer. 

Removal of the GST tag is often necessary to be able to perform functional or structural studies of the target protein. A specific protease site engineered between the GST moiety and the protein of interest allows removal of the GST moiety from the target recombinant protein. The GST can then be removed from the sample by re-chromatography on a glutathione column, and the protein of interest purified to homogeneity by other techniques such as gel filtration or ion exchange. 

Fusion proteins produced from a variety of E. coli pGEX vectors carry a PreScission Protease cleavage motif between the GST moiety and the cloned fusion partner. PreScission Protease is a genetically engineered fusion protein consisting of human rhinovirus 3C protease and GST. It specifically cleaves between the Gln and Gly residues of its recognition sequence of LeuGluValLeuPheGln/GlyPro.

PreScission Protease itself has a GST tag and therefore will bind to Glutathione Sepharose; it will thus not co-elute and contaminate the cleaved target protein. Cleavage with PreScission Protease is very specific, and maximum cleavage is obtained in the cold (the protein is most active at 5°C), thus improving the stability of the target protein. It can be used either following affinity purification or while fusion proteins are bound to Glutathione Sepharose columns. The molecular weight of PreScission Protease is approximately 46 kDa. 

 

 

 

Protocol: 


 

The following protocol describes the steps for cleavage of GST-fusion proteins with PreScission Protease: 

Note: The amount of PreScission Protease, temperature, and length of incubation required for complete digestion varies according to the specific GST-tagged protein produced. Optimal conditions should always be determined in pilot experiments. It is recommended that samples be removed at various time points and analyzed by SDS-PAGE to estimate the yield, purity, and extent of digestion during pilot experiments. 

PreScission Protease is provided at a concentration of 2U/μl (833 to 1000 U/mg) in Storage Buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 1 mM dithiothreitol, pH 8.0, 20% glycerol). Store the solution in small aliquots at -20°C in order to preserve activity. 

 

Unit Definition: One unit will cleave ≥ 90% of 100 μg of a test GST-fusion protein in Cleavage Buffer at 5°C for 16 hours.

 

Cleavage buffer preparation (before digestion): 50mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0 at 25°C. Chill to 5°C prior to use.

 

Note: Digestion may be improved by adding TritonTM X-100, TweenTM 20, NonidetTM, or NP40 to a concentration of 0.01%. Concentrations of these detergents up to 1% do not inhibit PreScission Protease activity. 

 

Prescission protease can be used for fusion protein cleavage in two ways:

1. On-column cleavage

2. Cleavage in solution 

 

On-column cleavage protocol: The removal of GST tags from target fusion proteins while they are still bound to the purification column is recommended as it facilitates cleavage of target protein and removal of the PreScission Protease from the sample in the same step. The fusion partner which has been cleaved from the GST moiety will be present in the flow-through whereas both PreScission Protease and the GST moiety will remain bound to the Glutathione Sepharose. Residual PreScission Protease remaining in the flow-through, if any, can be removed by passing the sample over fresh Glutathione Sepharose. 

 

  1. Bind the GST fusion protein sonicate to the appropriate bed volume of washed and equilibrated Glutathione Sepharose at 5°C. Bed volume is equal to 0.5X the volume of a 50% Glutathione Sepharose slurry used
  2. Wash the fusion protein-bound matrix with 10 bed volumes of Cleavage Buffer at   5°C and remove residual buffer.
  3. For each ml of washed Glutathione Sepharose bed volume, mix 40 μl (80 units) of PreScission Protease with 960 μl of Cleavage Buffer at 5°C. Add the PreScission Protease mixture to the fusion protein-bound Glutathione Sepharose and gently resuspend. Incubate at 5°C for 4 hours.  Note: More rapid cleavage may be achieved by adding a greater amount of PreScission Protease. 
  4. Collect eluate by either centrifugation of bulk Glutathione Sepharose matrix at 500 x g for 5 minutes or by collecting the eluate as it flows from the Glutathione Sepharose column. The eluate will contain the protein of interest, while the GST portion of the fusion protein and the PreScission Protease remain bound to the Glutathione Sepharose matrix.

 

Cleavage in solution: Following elution of the GST fusion protein from Glutathione Sepharose, the Prescission Protease can be used to remove the GST tag as follows.

 

  1. Dialyze the eluate, obtained following elution of the GST fusion protein from Glutathione Sepharose, extensively against Cleavage Buffer in order to remove reduced glutathione from the sample. Note: Alternatively, sample may be adjusted to 1X Cleavage Buffer by addition of the appropriate volume of 10X Cleavage Buffer. 
  2. Add 1 μl (2 units) of PreScission Protease for each 100 μg of fusion protein in the eluate. If the amount of fusion protein in the eluate has not been determined, add 40 μl (80 units) of PreScission Protease for each ml of Glutathione Sepharose bed volume from which the fusion protein was eluted. Incubate at 5°C for 4 hours. Bed volume is equal to 0.5X the volume of a 50% Glutathione Sepharose slurry used or 0.75X the volume of the original Glutathione Sepharose slurry supplied in the Bulk GST Purification Module. RediPack columns contain a 2 ml bed volume. MicroSpin columns have a 50 μl bed volume.  Note: More rapid cleavage may be achieved by adding a greater amount of PreScission Protease. 
  3. Once digestion is complete, apply the sample to washed and equilibrated Glutathione Sepharose to remove the GST portion of the fusion protein and the PreScission Protease from the protein of interest.

 

General PreScission Protease cleavage conditions: 

 

Component

Conditions

PreScission Protease

10 units/mg GST fusion protein

pH

7 - 8

dithiothreitol

1 mM

NaCl

0 - 500 mM

EDTA

1 - 10 mM

 

Reagents which inhibit PreScission Protease activity by > 50% at the concentrations indicated below: 

Reagent

Concentration

ZnCl2

100 mM

PefablocTM SC

4 mM

Chymostatin

100 mM

 

Reagents which fail to inhibit PreScission Protease activity by > 20% at the concentrations indicated below: 

Reagent

Concentration

Antipain dihydrochloride

74 mM

Aprotinin

0.3 mM

Bestatin

130 mM

E-64

28 mM

Leupeptin

1 mM

Pepstatin

1 mM

Phosphoramidon

0.6 mM

PMSF

1 mM

ZnCl2

10 mM

 


 

References:

 

  1. Harper S et al, Purification of proteins fused to glutathione S-transferase, Methods Mol Biol. 2011 ; 681: 259–280.
  2. Walker, P.A. et al., Biotechnology, 12, 601 (1994). 
  3. Cordingly, M.G. et al., J. Biol. Chem. 265, 9062 (1990).