Jul 01, 2020

Public workspaceTEV Protease V.1

This protocol is a draft, published without a DOI.
  • New England Biolabs1
  • 1New England Biolabs
  • New England Biolabs (NEB)
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External link: https://www.neb.com/protocols/2018/11/06/his-tag-removal-from-protein-using-tev-protease
Protocol Citation: New England Biolabs 2020. TEV Protease. protocols.io https://protocols.io/view/tev-protease-bfatjien
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 19, 2020
Last Modified: July 01, 2020
Protocol Integer ID: 35891
Keywords: his-tag removal, TEV , TEV protease, P8112,
Abstract
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.
  • Removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins
  • Optimal activity and stability for up to 24 months
  • Active in a wide range of buffers; optimal activity between pH 6.0 and 9.0.
  • High substrate specificity with no non-specific proteolysis

TEV Protease (NEB# P8112) is recommended for cleavage of a His-tag following purification with NEBExpress® Ni-NTA Magnetic Beads, Ni Spin Columns or Ni Resin. First, the expression vector must be designed to contain a TEV site between the His-tag and the protein.
Guidelines
Notes:
  1. If the fusion protein sample contains >2 M urea, >0.5 M Guanidine hydrochloride, >50 mM imidazole, pH values below 6 or above 9, or cysteine protease inhibitors then it will be necessary to dialyze the fusion protein into TEV protease reaction buffer before TEV Protease cleavage.
  2. TEV protease is inhibited by reaction buffers containing >40% Glycerol.
  3. Inhibition occurs in the presence of ≥ 5 mM Zn2+, ≥ 1 mM Cu2+and ≥ 10 mM Co2+.
  4. Compatible with 10mM MgSO4, MnCl2and CaCl2and up to 100mM EDTA.
  5. Compatible with the following protease inhibitors: aprotinin, benzamidine, leupeptin, pepstatin, PMSF.
  6. Optimal activity achieved in ≤0.2M NaCl; however, the enzyme retains some activity in up to 2M NaCl.
  7. Some substrates may require extended incubation periods (up to three days at either 4°C or 30°C) to achieve complete cleavage. The addition of more TEV Protease after 24 hours may also help achieve complete cleavage of some substrates.
Materials
MATERIALS
ReagentTEV proteaseNew England BiolabsCatalog #P8112S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Reactions may be scaled-up linearly to accommodate larger sample amounts and reaction volumes. Typical reaction conditions are as follows:
Typical Reaction Conditions for TEV Protease
Typical Reaction Conditions for TEV Protease
Dialyze the protein against Concentration20 millimolar (mM) Tris-HCl,, Ph7.5..
Determine the protein concentration.
Analyze
Combine Amount15 µg of protein and H20 (if necessary) to make a Amount45 µL total reaction volume.

Pipetting
Add Amount5 µL TEV Protease Reaction Buffer (10X) to make a Amount50 µL total reaction volume.

Pipetting
Add Amount1 µL TEV Protease .

Pipetting
Incubate at Temperature30 °C for Duration01:00:00 or at Temperature4 °C DurationOvernight .

Incubation