Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
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We have developed an algorithm (MassDynSearch) for identifying proteins using a combination of peptide masses with small associated sequences (tags). Unlike the approach developed by Matthias Mann, 'Tag searching', in which the sequence tags are generated by gas phase fragmentation of peptides in a mass spectrometer, 'Rag Tag' searching uses peptide tags which are generated enzymatically or chemically. The protein is digested either chemically or with an endopeptidase and the resultant mixture is then subjected to partial exopeptidase degradation. The mixture is analyzed by matrix assisted laser desorption and ionization time of flight mass spectrometry and a list of intact peptide masses is generated, each associated with a set of degradation product masses which serve as unique tags. These 'tagged masses' are used as the input to an algorithm we have written, MassDynSearch, which searches protein and DNA databases for proteins which contain similar tagged motifs. The method is simple, rapid and can be fully automated. The main advantage of this approach is that the specificity of the initial digestion is unimportant since multiple peptides with tags are used to search the database. This is especially useful for proteins like membrane, cytoskeletal, and other proteins where specific endopeptidases are less efficient and lower specificity proteases such as chymotrypsin, pepsin, and elastase must be used.
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PMID:An algorithm for the identification of proteins using peptides with ragged N- or C-termini generated by sequential endo- and exopeptidase digestions. 974 53

Two cDNA clones obtained from the Neospora caninum Expressed Sequence Tag project were selected by their homology with the Toxoplasma gondii serine proteinase inhibitor (serpin) gene, TgPI-1 and TgPI-2. One of them, named NcPI-H, showed several premature stop codons. The other cDNA, named NcPI-S, encoded a 79 amino acid protein containing a putative signal peptide and only one non-classical Kazal domain. Two other N. caninum EST sequences (NcEST1 and NcEST2) and one from Eimeria tenella (EtPI-S) were retrieved from the database. Amino acid sequence analysis suggested that NcEST1 and NcEST2 might be the N. caninum counterparts of TgPI-1 and TgPI-2, respectively. EtEST-S, as NcPI-S, is a single domain serpin. The open reading frame encoding the mature version of NcPI-S was expressed as recombinant protein, fused to a 6 histidine tag in Escherichia coli. Specific rabbit antiserum generated against the recombinant NcPI-S was used in immunoblot assays. Bands of 20, 30, 40, and 66-kDa were detected by SDS-PAGE of whole parasite homogenate. In addition, when an anti-TgPI-1 serum was used, bands of 25 and 35-kDa were detected indicating that there is no cross-reactivity between both serpins, and showing as well, the presence of another putative serpin in N. caninum. The recombinant protein NcPI-S, inhibited bacterial subtilisin completely, and showed lower inhibitory capacity on human neutrophil elastase, animal trypsin, and chymotrypsin, suggesting differences in effectiveness.
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PMID:Identification and characterization of serine proteinase inhibitors from Neospora caninum. 1513 71