Action
• Recombinant Enterokinase cuts the following sequence
Asp-Asp-Asp-Asp-Lys - X - X - X - X
• It has minimal requirements for specific amino acids in the P1’ and P4’ positions*
* Natural substrates have a propensity for Gly or Ser at P3’ and/or P4’ to ensure regular secondary
structure formation does not limit accessibility to the cleavage site.
• This independence makes enterokinase ideal for the removal of fusion proteins and/or tags
with the subsequent generation of an authentic N-terminus.
• Many companies monitor activity by following the cleavage of a protein. The fusion partners used
vary widely.
• The assay conditions employed also vary widely and so, the units of activity are often defined
differently.
• Paras uses one of the most credible industry standard methods for recording the activity of the
enzyme.
Recombinant Enterokinase
What
is Protein Purification
What
are Protein Tags?
Protein tags are peptide sequences genetically grafted
onto a recombinant protein which are often removable by
chemical agents or by enzymatic means. One such example
are affinity tags which are appended to proteins so that
they can be purified from their crude biological source
using an affinity technique.
Enterokinase can be used to cleave a fusion protein con-
taining a C-terminal affinity tag to produce a target protein
following protein purification.
Protein purification is a series of processes intended to
isolate one or more proteins from a complex structure. It is
vital for the characterisation of the function, structure and
interactions of the protein of interest.
One effective technique is the tagging of proteins to engi-
neer an antigen peptide tag onto a protein, and then purify
the protein through a column. Once finished, the tag can
be cleaved from the protein by a protease.
Enterokinase is a type 2 transmembrane serine protease produced by cells in the duodenum which are
involved in the body’s digestive system. It is responsible for the initial activation of pancreatic proteo-
lytic proenzymes that catalyses the conversion of trypsinogen to trypsin. Enterokinase’s specificity
makes it an ideal tool for biotechnological and biochemical removal of fusion proteins and tags.
Molecular weight of enterokinase varies from 82 - 140 kD (113 kD protein core) and 35 - 62 kD for its
disulfide-linked heavy-chain and light-chain, respectively, depending on the organism.
As a human enzyme, enterokinase is
1019 amino acids in length containing
14 disulfide bonds, 18 potential N-gly-
cosylation sites and a N-myristoyl lipda-
tion site as the N-terminus.
Enterokinase’s catalytic light chain is
235 amino acids in length and con-
tains 4 disulfide bonds.
Enterokinase deficiency is a life threat-
ening intestinal malabsorption disor-
der.
Specifically cleaves the bond between
Lys23 and lle24 in human trypsin-1
and the equivalent peptide bond in
other trypsin family members.
DISCOVERED BY NOBEL PRIZE WINNER
IVAN
PAVLOV
, IT IS THE FIRST KNOWN
ENZYME
TO
ACTIVATE OTHER ENZYMES.