Enterokinase Cleavage Enzyme (Mammalian Produced)
EK1 is a highly pure and active endotoxin-free enterokinase that is produced using human cell line in serum free media. It is highly specific for cleaving fusion proteins with the recognition sequence, Asp-Asp-Asp-Asp-Lys (DDDDK)
Because it is produced using human cell line, it has the appropriate glycosylation to catalyze highly accurate cleavage reaction with minimum non-specific activity. As seen below on lane 10, it provides almost complete reaction even at 0.25 unit of EK1, and zero non-specific cleavage of the substrate protein is seen even in the presence of 20 units of EK1 (seen under lane 2 below).
Its activity is stable over a broad temperature range, and it completes the reaction from 4°C to 60°C.
Unlike other site-specific proteases that cut within the recognition sequences leaving extra amino acids in the cleaved peptide products, the EK1 cleaves after the C-terminal end of the lysine residue, and the fragment produced from the cleavage reaction does not inherent any residues from the DDDDK recognition sequence. Therefore, the application can be extremely advantageous for producing a 100% native protein sequence and structure from recombinant fusion protein, which has the desired product immediately after the enterokinase recognition sequence, DDDDK. Owing to the presence of a His tag , EK1 can be easily removed after the cleavage reaction by affinity chromatography with Ni-IDA Agarose Beads.
In addition, DDDDK is a part of the octapeptide FLAG tag (DYKDDDDK), which can be utilized as a fusion tag for recognition by antibody, and for detection of fusion protein expression with Western blot analysis, as well as for purification of the fusion protein by Anti-FLAG affinity chromatography. This array of applications makes EK1 an ideal tool in the research involving the study of protein structure and function, and protein production where native protein structures and sequences are desired.
Enterokinase is used for site-specific cleavage of recombinant fusion proteins containing an accessible enterokinase recognition site for removal of affinity tags.
We recommend that the vial be briefly centrifuged prior to opening to bring the contents to the bottom. The stock solutions should be distributed into working aliquots and store at -20°C. Avoid multiple freeze/thaw cycles. Stable for 3 years from the date of shipping when stored and handled as per instructions.
|Unit quantity||100 Units|
|Does the enzyme have any tags?|
The enzyme is His-tagged.
|What is the pI value of the recombinant enterokinase?|
The calculated pI for our enterokinase is 5.2. The enzyme is rich in aspartate and glutamate and hence it is highly negatively charged. It binds to Q-sepharose columns at pH 7.
|How much can I a cut with one kit?|
How the protein will react upon enterokinase treatment is dependent on the structure of each individual protein, as well as the buffer condition. Thus, it must be evaluated and optimized by end user. From our experience on some proteins we have, 1 kit (100ul) will cleave about 1mg of protein.
|If the enterokinase is his-tagged, can I remove it using a metal-ion affinity column (such as nickel or cobalt)?|
Yes, this method can be used to remove enterokinase following the cleavage reaction.
|What is the % purity of this cleavage enzyme?|
The purity is >95%
|Can you tell me the concentration of enzyme provided?|
The approximate protein concentration of Enterokinase G699 is 10ug/1ml.
|What is the source of this enzyme?|
This enzyme has been produced from a mammalian cell expression system
|It can cleave at N-terminal end?|
It is advisable to place the tag at the N-terminus of your protein in order to remove the recognition site along with the His Tag in full. The Enterokinase will also be able to cleave the tag if it is placed at the c-terminus, however please be aware that this will result in the recogition sequence being left intact on your protein of interest, even after the his tag has been removed.
|Is it necessary to inactivate EK after cleavage?|
It is not generally necessary to deactivate the EK enzyme, however to prevent further degradation of the target protein after the reaction is finished, removal of enterokinase via affinity chromatography is recommended.
|What is an Enzyme Unit defined as?|
One unit is defined as the amount of TEV Protease that is required to cleave >90% of 3 µg of control substrate in a 30 µl reaction for 1 hour at 30°C in 1X TEV Protease Reaction Buffer supplemented with 1 mM DTT.
|What buffer is the enzyme supplied with?|
Enzyme supplied with 20X Reaction Buffer.
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