Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis.
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.
Kupke
T
T
Mikrobielle Genetik, Universität Tübingen, FRG.
Stevanovic
S
S
Ottenwälder
B
B
Metzger
J W
JW
Jung
G
G
Götz
F
F
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001430:Bacteriocins; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010455:Peptides; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000235:genetics; D011498:Protein Precursors / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012697:Serine Endopeptidases; D013212:Staphylococcus epidermidis / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; C098712:EpiA protein, Staphylococcus epidermidis; D008565:Membrane Proteins; D010455:Peptides; D010456:Peptides, Cyclic; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C050558:epidermin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase