Abstract
For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R-1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A(-4)-E-P-R(-1)- to -A(-4)-E-P-Q(-1)- by site-directed mutagenesis.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Anti-Bacterial Agents / biosynthesis
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Bacterial Proteins / genetics
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Bacterial Proteins / isolation & purification
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Bacterial Proteins / metabolism*
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Bacteriocins
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Endopeptidases / metabolism
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Escherichia coli / genetics
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Genes, Bacterial
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Membrane Proteins*
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Molecular Sequence Data
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Peptides*
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Peptides, Cyclic / biosynthesis
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Peptides, Cyclic / genetics
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Protein Precursors / biosynthesis
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Protein Processing, Post-Translational
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Protein Sorting Signals / genetics
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Protein Sorting Signals / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Serine Endopeptidases*
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Staphylococcus epidermidis / genetics
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Staphylococcus epidermidis / metabolism
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Substrate Specificity
Substances
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Anti-Bacterial Agents
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Bacterial Proteins
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Bacteriocins
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EpiA protein, Staphylococcus epidermidis
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Membrane Proteins
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Peptides
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Peptides, Cyclic
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Protein Precursors
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Protein Sorting Signals
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Recombinant Fusion Proteins
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epidermin
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Endopeptidases
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Serine Endopeptidases
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type I signal peptidase