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

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.
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PMID:Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum. 45 24

A method--enzymoblotting--was developed for localizing various enzymes after electrophoretic separation, transfer to nitrocellulose, and incubation with specific substrates. As an application, the proteinases porcine trypsin (EC 3.4.21.4), bovine chymotrypsin (EC 3.4.21.1), porcine elastase (EC 3.4.22.11), and their zymogen forms from porcine pancreas homogenate were analyzed utilizing specific p-nitroanilide substrates. After agarose gel electrophoresis, transfer of the separated proteinases to a nitrocellulose membrane was performed by capillary diffusion for 30 min. After air-drying of the nitrocellulose membrane, it was incubated in the appropriate substrate solution for 60 min. N-alpha-Benzoyl-DL-arginine-para-nitroanilide HCl was used as a substrate for trypsin, N-benzoyl-L-tyrosine-para-nitroanilide and succinyl-L-phenylalanine-para-nitroanilide for chymotrypsin, and N-succinyl-L-alanyl-L-alanyl-L-alanine-para-nitroanilide for elastase. p-Nitroaniline, the product thus obtained, was diazotized with N-(1-naphthyl)ethylenediamine to a red azo dye, visible at the site of the proteinases on the nitrocellulose membrane. The results could be preserved at -18 degrees C. Zymogen forms of the pancreas proteinases were detected in a similar manner. They were converted to active proteinases in situ on the nitrocellulose membrane after preincubating the nitrocellulose membrane in the activation enzymes enteropeptidase or trypsin.
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PMID:Enzymoblotting: a method for localizing proteinases and their zymogens using para-nitroanilide substrates after agarose gel electrophoresis and transfer to nitrocellulose. 351 6

Human granulocyte colony stimulating factor (hG-CSF) was expressed in the methylotrophic yeast Pichia pastoris, using two different constructs which resulted in proteins with different N-terminal sequences. In the first construct, a hexa-histidine tag and enterokinase cleavage site were added to the N-terminus of the protein to achieve one-step separation and exact processing. In the second construct, the gene was fused to the alpha-MF prepro leader at the Lys-Arg processing site (without Glu-Ala spacer). The PCR products were cloned in pPIC9 commercial vector and integrated into the alcohol oxidase region of the host genome. Transformation was done by electroporation or spheroplasting. Selection of good producing clones was performed by immunoblot analyses of the supernatants from shake-flask fermentation. Proper processing of the products was confirmed by N-terminal sequencing of the secreted proteins. With both plasmid constructs, the target proteins, bearing the histidine tag or not, represented majority of the secreted proteins. Although the proteins were present in the soluble form, they were highly aggregated, which interfered with purification. The most efficient way to obtain monomeric, biologically active protein was complete denaturation by guanidine-HCl or urea and subsequent renaturation during gel filtration chromatography.
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PMID:Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast pichia pastoris. 1167 33

The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)(6)-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8M urea. Use of a multi-component screen identified the optimal conditions for folding (His)(6)-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3(-) CD8+ lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography.
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PMID:E. coli expression and purification of human and cynomolgus IL-15. 1943 2