May 18, 2020

Public workspaceCleavage of the Fusion Protein (TEV Protease)

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  • New England Biolabs (NEB)
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Protocol CitationNew England Biolabs 2020. Cleavage of the Fusion Protein (TEV Protease). protocols.io https://dx.doi.org/10.17504/protocols.io.bfd9ji96
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 21, 2020
Last Modified: May 18, 2020
Protocol Integer ID: 36001
Keywords: TEV, protease, cleavage,
Abstract
TEV Protease, also known as Tobacco Etch Virus (TEV) Protease, is a highly specific cysteine protease that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues. It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins. TEV Protease has a 7xHis-tag for easy removal from a reaction using nickel affinity resins and has been engineered to improve thermal stability and decrease autolysis. 
Guidelines
Affinity tags, such as Maltose Binding Protein (MBP) or polyhistidine (His), are essential tools for the production of recombinant proteins. MBP is known to significantly enhance the solubility of many proteins, resulting in higher yield. Whereas, His-tagging is widely employed for the purification of recombinant target proteins via immobilized metal affinity chromatography (IMAC). The His-tag advantages include small tag size and high affinity and specificity of poly-His tag binding to divalent metals at neutral pH.  Despite these important advantages, it is often preferred to remove the affinity tag following purification in order to isolate the target protein. Although chemical and enzymatic methods can be used, proteolytic enzymes, such as TEV Protease (NEB #P8112) and Factor Xa (NEB #P8010) are the preferred method for cleavage of fusion proteins at designed cleavage sites.
Materials
MATERIALS
ReagentTEV proteaseNew England BiolabsCatalog #P8112S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
MBP and the target protein are fused by a polylinker containing a TEV protease recognition site for easy removal of the MBP-tag. One unit of TEV Protease will cleave approximately 2 μg of fusion protein. Cleavage should be carried out in 1X TEV Protease Reaction Buffer or in Amylose column elution buffer supplemented with DTT to a final concentration of 1 mM. Depending on the particular fusion protein, the amount of TEV Protease can be adjusted to get an acceptable rate of cleavage.
If necessary, concentrate the fusion protein to at least Concentration0.5 milligram per milliliter (mg/mL) .

Perform a pilot experiment with a small portion of your protein. For example:
Combine Amount15 µg fusion protein and H2O to make a total reaction volume of 45 μl.

Pipetting
Add Amount5 µL TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.

Pipetting
Add Amount1 µL TEV Protease .

Pipetting
In a separate tube, combine Amount5 µg fusion protein , Amount5 µL TEV Protease Reaction Buffer (10X) and H2O to a volume of 50 μl. Do not add TEV Protease (control sample).

Pipetting
Incubate reaction and control sample for Duration01:00:00 , Duration03:00:00 , and Duration08:00:00 at Temperature30 °C (an additional reaction can be made and incubated for 24 hours at 4°C).

Incubation
Take Amount10 µL of reaction(s) at the indicated times above and add Amount5 µL SDS-PAGE Sample Buffer (3X) . Take Amount10 µL control sample and add Amount5 µL SDS-PAGE Sample Buffer (3X) after 8 hours (or longest incubation time).

Pipetting
Incubate the SDS-PAGE samples for Duration00:03:00 -Duration00:05:00 at Temperature70 °C -Temperature100 °C .

Incubation
Analyze them by SDS-PAGE.
Analyze
Scale up the pilot experiment linearly for the amount of the fusion protein to be cleaved. Save at least a small sample of the uncut fusion as a negative control.
Check for complete cleavage by SDS-PAGE.
Analyze
TEV Protease and the cleaved MBP contain polyhistidine tags at their N-termini. They can be removed from the cleavage reaction by immobilized metal affinity chromatography, such as Nickel or Cobalt resin, thereby isolating the target protein in the flow through.
Optional