CleaTag™ Bovine Enterokinase

Overview

Description

CleaTag™ Recombinant Bovine Enterokinase (EK,his-tagged)

Tested applications

Fusion tag removal from the recombinant peptide fragment or protein that is preceded by the characteristic sequence DDDDK

 

Cleavage or processing of engineered proteins that contain enzyme recognition sequence.

 

Processing enzymes and proteins that are unstable in room temperature

 

Enterokinase will not work if the recognition site is followed by a proline.

 

rbovine EK with 6 × His-tag binds with Ni2+ affinity resin and was designed for removing from digestion system.

Properties

Storage instruction

The product is shipped with blue ice. Store at -20°C upon receipt. It remains stable without detectable loss of activity after one week at 25°C. Stable after several freeze-thaw cycles.

Database links

Uniprot：P98072 (ENTK_BOVIN)

 

A DNA sequence encoding the bovine TMPRSS15 (NP_776864.1) (Ile801-His1035) was expressed with a polyhistidine tag at the C-terminus.

CAS #

9014-74-8

Synonyms

Enteropeptidase, EC 3.4.21.9, Enterokinase Light Chain, Serine protease 7

Molecular weight

Recombinant Bovine Enterokinase is a single glycosylated polypeptide chain containing 235 amino acids and 6 histidines, so predicts a molecular mass of 27.1 kDa.

Specific activity

≥5 U/μL

Unit definition

One unit is defined as the amount of enzyme needed to cleave 0.5 mg of a special fusion protein in 12 to 16 hours to get 95% completion at 25°C in a buffer containing 25 mM Tris-HCl (pH 8.0).

Enzyme Commission #

3.4.21.9

Subunit

Enterokinase (EK) is an amino protease existing in duodenum of mammal and is involved in digestion. It consists of a disulfide-linked 82–140 kDa heavy chain which anchors enterokinase in the intestinal brush border membrane and a 35–62 kDa light chain which contains the catalytic subunit. Additionally, both of the chains are derived from a single precursor that is cleaved by a trypsin-like protease. EK can specially recognize the amino acid sequence DDDDK, and digest the peptide bond after the lysine residue. rEK was report to be more effective than nature EK in cleaving recombinant proteins. Furthermore, the light chain possesses the whole enzyme activity of EK. rBoEK has the highest activity than EK of other species and is used wildly in biochemical applications.

Endotoxin Level

< 1.0 EU/μg, determined by LAL method.

Purity

> 97% as analyzed by SDS-PAGE.

Applications

Application Image

Recombinant bovine enterokinase shows optimum cleavage activity at 25°C, without detectable nonspecific enzymatic activities (Figure 1). When incubated for 24 hours at 25°C, 0.1 U of EK is sufficient for complete digestion of 50 µg substrate. When enzymatic reaction was extended to 64 hours, only one protein fragment of the expected product size was revealed, suggesting no cleavage outside of the specific enzyme-recognition sequence.  

Figure 1. Optimum EK cleavage reaction occurs at 25°C. 

A substrate (50 µg) was incubated with 0.1 U enterokinase at 25°C for extended period of time, from 8 hours to 64 hours. The resulted protein fragmants after enzymatic reactions were analyzed by SDS-PAGE. Incubation of the substrate with 0.1 U of EK for longer period resulted digestion to completion, without nonspecific cleavage. No degradation of the digested substrate was detected.

Highlights

High activity

One unit is able to cleave 0.5 mg of a DDDDK-containing protein to 95% completion in 12 to 16 hours at 25°C. It is supplied as concentrated solution of > 5U/µL.

 

High purity

There are no any other protease contaminants nor animal-derived impurities.

 

High specificity

1. Protease that cleaves specifically after a lysine preceded by four aspartic acids: Asp-Asp-Asp-Asp-Lys (DDDDK)

2. No detectable off-target cleavage when used at 2 - 25°C.

Long-lasting superior performance. Retained enzymatic activity at 2 - 8°C.

It allows low-temperature digestion to ensure stability of temperature-sensitive target proteins

 

Compatible with the following reagents:

Imidazole (<100 mM), NaCl (<50 mM), and glycerin (<5%).

Application Figure 1

Protocols

Recommendations for fusion protein digestion

EK Dilution/Storage Buffer: 20mM Tris-HCl, pH 7.4, 200mM NaCl, 2mM CaCl₂, 50% glycerol

Cleavage Reaction buffer: 25mM Tris-HCl, pH8.0

Fusion protein concentration: 0.1 ~ 1 mg/ml (total protein content: 0.5-1.0 mg)

EK content: 1-2 U per reaction

Temperature: 25°C

Incubation time: 12h-16h or overnight
 
Attention

Certain chemicals and reagents may affect Enterokinase activity, including:

 

Imidazole (>200 mM), NaCl (>200 mM), and glycerin (>5%)

 

It is necessary to decrease the concentration of agents that may interfere enterokinase activity prior to enzyme digestion. Proceed with one of the followings:

 

• To achieve optimum result, dialyze the sample against 25 mM Tris-HCl (pH 8.0) before digestion;
• If dialysis is undesirable, dilute the sample to a final solution that contains <100 mM imidazole, <50 mM NaCl, and <5% glycerin. Maintain the ratio of EK to fusion protein at 1 U for every 0.5 mg fusion protein in the reaction;
• If the sample solution contains one or more components which concentrations are higher but further dilution or depletion is not desirable, it is also recommended to increase the EK amount in the reaction and to extend the reaction time.