Rules of Coexistence: RiPP Recognition Elements Evolved to Prevent Pathway Interference through Leader Peptide Discrimination

Identification of the precursor peptide post translational modifications by MALDI-TOF MS followed by Mascot search.
LppK kinase modifies conserved Ser3 residue in LppA. LppA peptide fused with N-terminal MBP and C-terminal TrxA mass tag (MW 60.1 kDa) co-expressed with LppB harboring N-terminal Strep-tag and LppK. The LppB-LppA complex was purified using affinity chromatography. Protein zones corresponding to LppA (MBP-LppA-TrxA fused protein, MW 61.5 kDa) were subjected to in-gel digestion with trypsin. The eluted tryptic peptides were identified using MALDI-TOF MS.
For the leader peptide cleavage site identification, LppA peptide fused with N-terminal MBP and C-terminal TrxA mass tag (MW 60.1 kDa) co-expressed with LppB harboring N-terminal Strep-tag and LppB2. The LppB-LppA complex was purified using affinity chromatography. Protein zones corresponding to LppA (MBP-LppA-TrxA fused protein, MW 61.5 kDa) were subjected to in-gel digestion with trypsin. The eluted tryptic peptides were identified using MALDI-TOF MS.