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TEV Cleavage Protocol for Removal of C-terminal STREP and DET Tags

Introduction​

This protocol describes a TEV cleavage approach for removing the STREP and DET tags from the C-terminus of proteins produced using the eProtein™ Discovery Scale-up Expression and Purification guide. This protocol is designed to support users working with proteins where C-terminal tags may interfere with biological function, structural integrity, or downstream applications. By removing these tags through TEV protease cleavage, users can obtain a more native-like version of the protein for functional assays, binding studies, or structural characterization.

This protocol is designed to support users working with proteins where C-terminal tags may interfere with biological function, structural integrity, or downstream applications. By removing these tags through TEV protease cleavage, users can obtain a more native-like version of the protein for functional assays, binding studies, or structural characterization

Note: Following TEV cleavage, the protein will lack the STREP and DET tags for purification and quantification with the eProtein™ Quantification kit. The resulting protein will retain six non-native amino acids (ENLYFQ) from the TEV recognition sequence.

IMPORTANT

This protocol does not cover the removal of TEV protease. Please consult manufacturer guidelines if TEV protease removal is required for downstream applications.

TEV cleavage​

Material Required​

Item
Eluted protein scale-up
TEV protease stored in 1 mM TCEP (e.g. NEB® P8112S or MERCK® T4455)
SDS-PAGE equipment (for analysis)

Workflow Schematic​

Figure 1. TEV cleavage workflow schematic. The final sample will contain the cleaved protein, detached C-terminal DET and STREP tags, TEV protease, and potentially some uncleaved protein if cleavage efficiency is suboptimal.

Protocol​

  1. Express and purify the protein according to the standard eProtein™ Discovery Scale-up Expression and Purification guide.
  2. Reserve 3 µL of the uncleaved sample for SDS-PAGE analysis.
  3. Dilute TEV protease 1:20 ratio into the eluted protein. Example: Add 13 µL TEV to 250 µL of eluted protein. No additional buffers or reducing agents are required.
  4. Gently pipette to mix and distribute the TEV protease.
  5. Incubate at 4°C for 18 hours.
  6. Optional: If required, remove TEV protease using the manufacturer’s protocol.
  7. Verify cleavage by SDS-PAGE against uncleaved samples from Step 2.

Figure 2. Example SDS-PAGE of successful TEV cleavage. “–” indicates the uncleaved sample while “+” represents the TEV-cleaved products.

 NEB is a registered trademark of New England Biolabs, Inc. Merck is a registered trademark of Merck KGaA and Merck & Co., Inc. Product codes and catalog numbers are provided for reference only and may vary between regions or distributors. Please check with your local supplier for availability and compatibility.



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