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)

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His-tagged ZntR and purified by Ni2+-affinity column. His-tag-free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 x 10-12 M. The addition of 25 microM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus, to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription from the znt promoter in a concentration-dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans-acting repressor protein that binds to the znt promoter region and regulates its own transcription together with that of zntA.
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PMID:ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus. 1041 36

Granzyme B is a cytotoxic lymphocyte granule serine proteinase that is pivotal in the induction of target cell apoptosis. Here we describe the expression of recombinant human granzyme B in Pichia pastoris as a chimeric zymogen comprising the alpha-factor signal sequence, a prodomain including an enterokinase cleavage site, and the mature granzyme B sequence followed by a hexahistidine tag. Inactive zymogen is purified from the medium by immobilized cobalt chelate affinity chromatography and then activated by enterokinase (final yield is approximately 1 mg per liter). The recombinant enzyme resembles native granzyme B in size and glycosylation, hydrolyzes the substrate Boc-Ala-Ala-Asp-thiobenzyl ester with equivalent efficiency (K(m) 82 microM; k(cat) 12 s(-1)), processes procaspase-3 to subunit form, and is inhibited by the cognate serpin PI-9. It efficiently induces DNA degradation and apoptosis of human cells. The availability of recombinant human granzyme B will facilitate further investigation of its structure and role in immune effector cells.
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PMID:Expression and purification of recombinant human granzyme B from Pichia pastoris. 1042 74

The extracellular loop 2 of a tetraspanin from Schistosoma japonicum (Sj-TSP-2) is homologous to Schistosoma mansoni TSP-2. In our initial study, Sj-TSP-2 is an identical antigen against schistosomiasis caused by S. japonicum. Through the pET32 vector system and nickel (Ni)-absorbed chelating Sepharose, Sj-TSP-2 was expressed and purified as a soluble fusion constructed with an N-terminal thioredoxin-His(6)-EK protease site tag (Trx-TSP-2). In phosphate buffer (PB) with a low concentration of imidazole, the Trx-TSP-2 fusion protein was efficiently cleaved by enterokinase (EK). Sj-TSP-2 was isolated and enriched using cobalt (Co)-absorbed chelating Sepharose and HiTrap SP column. Character of the protein was analyzed via animal experiments and then clinical trials. The purification approach yielded pure Sj-TSP-2, which will provide feasible advices for discovering vaccines against schistosomiasis.
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PMID:Schistosoma japonicum: efficient and rapid purification of the tetraspanin extracellular loop 2, a potential protective antigen against schistosomiasis in mammalian. 2068 2