Project description:A previously reported gene cluster encoding four nisin-like peptides, three with the same sequence (NsoA1-3) and the unique NsoA4, produced antimicrobial activity in the presence of trypsin after heterologous expression in Lactococcus lactis. Protein extracts were prepared by SDS gel electrophoresis or immunoprecipitation using an antibody to the NsoA2 leader. The tryptic peptides observed by LC-MS/MS covered the complete sequence of preNsoA1-3 and part of the leader sequence of preNsoA4 and confirmed the expression and the predicted sequences of the preNsoA peptides. Further, the data revealed that the preNsoA1-3 peptides were partly modified with dehydrations and formation of lanthionine rings. A certain amount of fully modified preNsoA1-3 was observed. Details of modifications of the core peptide and the C-terminal tryptic peptide TATCGCHITGK covering rings D and E indicated that 22% of these preNsoA1-3 peptides were completely modified. A lower amount is estimated for rings A-C. Intact masses of immunoprecipitation derived peptides determined by LC-MS accurately matched the expected preNsoA precursor peptides. The most abundant peptides detected were preNsoA2-3-8H2O followed by preNsoA1-8H2O and other states of dehydration. The results indicate that processing occurs but is incomplete for preNsoA peptides in the heterologous system with the formation of a certain amount of fully modified and active peptides.
