Cleavage Enzymes for Fusion Protein Purification

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After purifying epitope-tagged fusion proteins using agarose or magnetic bead resins, you can use MilliporeSigma’s cleavage enzymes to isolate your untagged, intact protein of interest.

Use the table below as a guide to choosing the cleavage enzyme that matches your affinity purification scheme:

Cleavage Enzyme
Recognizes Sequence
Factor Xa Ile-(Glu or Asp)-Gly-Arg-X
Enterokinase Asp-Asp-Asp-Asp-Lys-X
Thrombin Leu-Val-Pro-Arg↓Gly-Ser
HRV3C Protease Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro
Discover our Cleavage Enzyme. Click on the cleavage enzymes below to get more information:

Factor Xa cleavage gel.
Factor Xa Cleavage: 10 µg of Factor Xa cleavage control protein (CCP, lane 1) was digested with increasing amounts (0.03 µg to 0.5 µg per reaction) of Factor Xa (lanes 2-5). SDS-PAGE (4-20% gradient gel) followed by staining with Coomassie® Blue showed the two expected proteolysis products (~35 kDa and ~17 kDa).

Factor Xa - Cleavage Enzymes

Factor Xa is an endopeptidase that cleaves proteins at the sequence Ile-(Glu or Asp)-Gly-Arg-X, where X is any amino acid other than proline or arginine. Factor Xa cleaves at the C-terminal end of the Arg residue, and is frequently used to remove polyhistidine tags during protein affinity purification on nickel columns.

The Factor Xa Cleavage Capture Kit is designed for highly specific cleavage of fusion proteins followed by convenient affinity-based capture and removal of Factor Xa. After cleavage of the target protein, Factor Xa is removed with greater than 99% efficiency from the reaction by affinity capture on Xarrest™ Agarose. Restriction Grade Factor Xa is a highly purified preparation isolated from bovine plasma and activated with Russell's viper venom. This preparation is purified to near homogeneity and shows no secondary cleavage from contaminating proteases.

Enterokinase - Cleavage Enzyme

Enterokinase specifically cleaves proteins at the specific sequence, Asp-Asp-Asp-Asp-Lys-X, with X being any amino acid other than proline, at the C-terminal end of the lysine. Since this sequence is included in the widely used FLAG affinity tag, Enterokinase is a commonly used enzyme for protein affinity purification.

The Enterokinase Cleavage Capture Kit and Tag•off™ rEK Cleavage Capture Kits are designed for highly specific cleavage of fusion proteins followed by rapid, affinity-based capture and removal of enterokinase.

Recombinant Enterokinase (rEK) is a highly purified preparation of the catalytic subunit of bovine enterokinase, a serine protease which recognizes the identical cleavage site as the native enzyme and has similar enzymatic activity.
SDS-PAGE of thrombin cleavage
Biotinylated Thrombin cleavage with respect to amount of thrombin added. The indicated amounts of biotinylated thrombin were used to cleave 2 μg of Cleavage Control Protein in an overnight digestion. Samples were analyzed by SDS-PAGE (4-20% gradient gel) followed by staining with Coomassie blue. The 0.0045-unit lane represents a 2.25-fold overdigestion.

Thrombin - Cleavage Enzyme

Restriction-grade and biotinylated thrombin enzymes are ideal for highly efficient cleavage of fusion proteins.

Restriction Grade Thrombin is qualified to specifically cleave target proteins containing the recognition sequence LeuValProArg↓GlySer. The preparation is functionally tested for activity with fusion proteins and is free of detectable contaminating proteases. Thrombin is supplied with 10X Thrombin Cleavage Buffer and a Cleavage Control Protein.

Biotinylated Thrombin is identical in activity to Restriction Grade Thrombin, but has covalently attached biotin for easy removal of the enzyme from cleavage reactions using immobilized streptavidin. Our Thrombin Cleavage Capture Kit not only includes biotinylated thrombin and immobilized streptavidin but also all required buffers and filters for complete, convenient recovery of cleaved protein.


HRV 3C protease cleavage vs. competitor
HRV 3C Protease cleaves fusion proteins more efficiently (lanes 2 and 4) compared to cleavage with a competitor’s protease (lanes 3 and 5).
Experimental Methods
Using a 1:100 (w/w) ratio of protease:target protein, 500 μg of purified Nus•Tag™ enolase fusion protein was incubated in parallel 500 μL reactions at 4°C. The reactions was quenched by adding equal volume 4X SDS Sample Buffer and then immediately placing the samples into a water bath at 75 °C for 5 min. Lane M - PerfectProtein Markers, 10-225 kDa, lane 1 - 3 μg purified Nus•Tag enolase fusion protein, lane 2 - 3 μg Nus•Tag enolase fusion protein with 30-min HRV3C protease reaction, lane 3 - 3 μg Nus•Tag enolase fusion protein with 30-min competitor’s protease reaction, lane 4 - 3 μg Nus•Tag enolase fusion protein with 60-min HRV3C protease reaction, lane 5 - 3 μg Nus•Tag enolase fusion protein with 60-min competitor’s protease reaction.

HRV 3C Protease – Cleavage Enzyme

Recombinant type 14 3C protease from human rhinovirus (HRV 3C) is a highly purified, recombinant 6XHis-tagged enzyme, which recognizes the cleavage site LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease, with optimal activity at 4 ºC, high specificity, and His-tag fusion make HRV 3C protease an ideal choice for rapid removal of fusion tags, even from labile target proteins.