Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.9 (enterokinase)
 675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a T7-based Escherichia coli expression vector in which protein coding sequence is seamlessly fused to the N-terminal calmodulin-binding peptide (CBP) purification tag. We combined the use of the site-specific protease enterokinase (EK) and the type IIs restriction enzyme Eam1104 I, which cleave outside their respective (amino acid and nucleotide) target sequences, such that any amino acid sequence may be fused directly C-terminal to the EK cleavage site without codon constraints conferred by the cloning method. PCR products are cloned using ligation-dependent or ligation-independent methods with high cloning efficiencies (>10(6) cfu/microg vector), allowing production of insert quantities sufficient for several cloning experiments with a limited number of PCR cycles, resulting in a significant time-savings and reduced likelihood of accumulating PCR-derived mutations. CBP fusion proteins are expressed to high levels when the CBP peptide is positioned at the N-terminus. CBP binds to calmodulin with nanomolar affinity, and fusion proteins are purified to near homogeneity from crude extracts with one pass through calmodulin affinity resin using gentle binding and elution conditions. We show high efficiency seamless cloning of three inserts into the pCAL-n-EK vector, including one encoding the protein c-Jun N-terminal kinase (JNK). CBP-EK-JNK fusion protein was synthesized to 10-20 mg/liter culture and purified to near homogeneity in one step with calmodulin affinity resin. The fusion tag was efficiently removed with EK to yield active JNK with native N-terminal amino acid sequence.
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PMID:An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions. 1033 54

Metabotropic glutamate receptors (mGluRs) influence a variety of second-messenger systems and ion channels. The C-terminal region of group III mGluRs interacts with the Ca(2+)-binding protein calmodulin (CaM). We intend to study the interaction between Ca(2+)/CaM and the CaM-binding motifs within mGluR(7), which is a group III mGluR. We established a recombinant protein expression and purification system for nuclear magnetic resonance (NMR) analysis of mGluR(7) peptides using Escherichia coli. Peptides of mGluR(7) conjugated to an affinity tag sequence were constructed, and protocols for expression and purification were optimized. To suppress non-specific enzymatic cleavage, the mGluR(7) fusion peptide was bound to Ca(2+)/CaM before enterokinase cleavage. This complex method for precise enzymatic reactions may be applicable for the recombinant preparation of a wide variety of peptides.
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PMID:Expression and purification of metabotropic glutamate receptor 7 peptides. 2036 28

Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags.
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PMID:Several affinity tags commonly used in chromatographic purification. 2449 Jan 6