Restriction Grade Thrombin and Biotinylated Thrombin

Thrombin, Restriction Grade

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.

Unit definition: one unit is defined as the amount of enzyme needed to cleave 1 mg of fusion protein in 16 hours at 20°C in a 200 µl reaction containing 20 mM TrisHCl pH 8.4, 150 mM NaCl, 2.5 mM CaCl2, 50 µg fusion protein and enzyme.

Biotinylated Thrombin

Biotinylated Thrombin is similar to Restriction Grade Thrombin, but has covalently attached biotin for easy removal of the enzyme from cleavage reactions using immobilized streptavidin. Novagen’s preparation is tested for activity using the same assay as for unmodified thrombin, and for greater than 99% binding to Streptavidin Agarose (Cat. No. 69203-3).

Biotinylated Thrombin cleavage

Biotinylated Thrombin cleavage. 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 units lane represents a 2.25-fold overdigestion.

Thrombin Cleavage Capture Kit

The Thrombin Cleavage Capture Kit is designed for cleavage of fusion proteins followed by convenient and quantitative removal of thrombin protease. The method is based on the use of Biotinylated Thrombin for digestion and its subsequent removal with Streptavidin Agarose. The kit is suitable for use with any fusion protein that contains a thrombin recognition sequence.

A Cleavage Control Protein is included in the kit to monitor performance of cleavage conditions. The 48 kDa control protein is cleaved by thrombin into two proteolytic fragments of 35 kDa and 13 kDa, which are easily visualized by SDS-PAGE.

Recombinant Enterokinase

Recombinant Enterokinase (rEK) is a highly purified preparation of the catalytic subunit of bovine enterokinase, which recognizes the identical cleavage site as the native enzyme (i.e., AspAspAspAspLys↓) and has similar enzymatic activity. rEK exhibits superior rates of cleavage of fusion proteins containing the recognition sequence when compared to the native enzyme.1 Novagen’s rEK is purified to near homogeneity and, unlike some preparations of native bovine enterokinase, exhibits no secondary cleavage arising from contaminating proteases. The preparation is also functionally tested for activity with fusion proteins.

Unit definition: one unit is defined as the amount of enzyme needed to cleave 50 µg of fusion protein in 16 hours at 23°C in a buffer containing 20 mM Tris-HCl pH 7.4, 50 mM NaCl, and 2 mM CaCl2.

Recombinant Enterokinase (rEK) cleavage

Recombinant Enterokinase (rEK) cleavage. The Cleavage Control Protein (3 µg) was digested with increasing amounts of rEK in separate reactions under standard assay conditions. Samples were analyzed by SDS-PAGE (4–20% gradient gel) followed by staining with Coomassie blue. The 0.06 units lane corresponds to 1 enzyme unit per 50 µg target protein, which exhibits > 95% cleavage.

Enterokinase Cleavage Capture Kit

The Enterokinase Cleavage Capture Kit is designed for highly specific cleavage of fusion proteins followed by rapid, affinity-based capture and removal of enterokinase.

Following cleavage of the target protein, rEK is removed with > 99% efficiency from the reaction by affinity capture on EKapture Agarose. Following capture of rEK, the EKapture Agarose is removed by spin filtration. Because the same buffer conditions are used for both cleavage and capture, no buffer changes are necessary.

The kit also includes a Cleavage Control Protein for conducting control digests in parallel with experimental samples, or to test cleavage under customized buffer conditions. The 48 kDa Cleavage Control Protein is cleaved by rEK into two proteolytic fragments of 32 kDa and 16 kDa, which are easily visualized by standard SDSPAGE followed by Coomassie blue staining (see figure below). The Cleavage Control Protein also features an amino terminal S•Tag sequence enabling sensitive detection of the 16 kDa proteolytic product with Western blot reagents.

Restriction Grade Factor Xa

Restriction Grade Factor Xa is a highly purified enzyme isolated from bovine plasma and activated with Russell’s viper venom. Novagen’s preparation is purified to near homogeneity and shows no secondary cleavage from contaminating proteases. The preparation is also functionally tested for activity with fusion proteins. Like enterokinase, Factor Xa cleaves at the C-terminal side of its recognition sequence (IleGluGlyArg↓) and can therefore be used for removing all vector-encoded sequences from appropriately designed constructs.

Unit definition: one unit of Restriction Grade Factor Xa cleaves 50 µg Xa Cleavage Control Protein to > 95% completion in 16 hours at 21°C in a buffer containing 50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 5 mM CaCl2.

 

Factor Xa cleavage

Factor Xa cleavage. The Xa Cleavage Control Protein (3 µg) was digested with increasing amounts of Factor Xa in separate reactions under standard assay conditions. Samples were analyzed by SDS-PAGE (4–20% gradient gel) followed by staining with Coomassie blue. The 0.015 units lane corresponds to 0.25 units enzyme per 50 µg target protein, which exhibits > 95% cleavage.

Factor Xa Cleavage Capture Kit

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 95% efficiency from the reaction by affinity capture on Xarrest™ Agarose. Following capture of Factor Xa, the agarose is removed by spin-filtration. No buffer changes are necessary because the same buffer conditions are used for both cleavage and capture. The kit also includes a Cleavage Control Protein for conducting control digests in parallel with experimental samples, or to test cleavage under customized buffer conditions. The 49 kDa Xa Cleavage Control Protein is cleaved into two proteolytic fragments of 32 kDa and 17 kDa, which are easily visualized by standard SDS-PAGE followed by Coomassie blue staining (see figure below). The Xa Cleavage Control Protein also features an amino terminal S•Tag sequence enabling sensitive detection of the 17 kDa proteolytic product with Western blot reagents.

 

Materials

Product No. Description
69069 Cleavage Control Protein
69671 Thrombin, Restriction Grade
69672 Biotinylated Thrombin
69022 Thrombin Cleavage Capture Kit
69203 Streptavidin Agarose
69072 Spin Filter, 2 ml
69066 Recombinant Enterokinase
69067 Enterokinase Cleavage Capture Kit
69068 EKapture™ Agarose
69036 Factor Xa, Restriction Grade, Bovine Plasma
69037 Factor Xa Cleavage Capture Kit
69038 Xarrest™ Agarose
69051 Xa Cleavage Control Protein

 

Reference

  1. Collins-Racie, L.A., et al. (1995) Bio/Technology 13, 982–987.