Open AccessPurification and characterization of EpiA, the peptide substrate for post‐translational modifications involved in epidermin biosynthesis
Author(s) -
Kupke Thomas,
Stevanovic Stefan,
Ottenwälder Birgit,
Metzger Jörg W.,
Jung Günther,
Götz Friedrich
Publication year - 1993
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1993.tb06421.x
Subject(s) - biosynthesis , lantibiotics , biochemistry , cleavage (geology) , escherichia coli , fusion protein , chemistry , peptide , amino acid , enzyme , substrate (aquarium) , peptide sequence , biology , bacteria , recombinant dna , gene , genetics , paleontology , ecology , fracture (geology) , nisin
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.