Fig. 2: In vitro expression, modification, and maturation of salivaricin B in UniBioCat.
From: Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides
a The gene cluster (top) and catalytic steps (bottom) of salivaricin B biosynthesis. Dha, dehydroalanine; Dhb, dehydrobutyrine; Abu, α-aminobutyric acid. b Western-blot analysis of the cell-free expressed precursor peptide (left: SboA, 7.8 kDa) and modification enzymes [right: SboM (113 kDa) and SboT (83 kDa) are co-expressed]. Peptides/enzymes are labeled with the anti-His antibody. M, protein marker; T, total protein; S, soluble protein. Results were reproduced three times independently; representative data are shown. c MALDI-TOF-MS analysis of the precursor peptide (left: 6x His-tagged SboA, [M + H]+ m/z = 7355) and modified precursor peptides (right: dehydrated 6x His-tagged SboA, [M-H2O + H]+ m/z = 7337, [M-2H2O + H]+ m/z = 7319, and [M−4H2O + H]+ m/z = 7283). d MALDI-TOF-MS (left), LC-MS (middle), and MS/MS (right) analysis of matured salivaricin B. e MALDI-TOF-MS (left), LC-MS (middle), and MS/MS (right) analysis of the analog salivaricin B−1 with one additional amino acid alanine at the N-terminus. f Antimicrobial activity assay of salivaricin B (SalB) and salivaricin B-1 (SalB-1). “SalB & B-1” was cell-free synthesized and purified for the activity assay (note that the sample used was a mixture of SalB and B-1 with a concentration of 1 mM for the test). For in vivo produced SalB and SalB-1, 1 mM of each purified SalB and SalB-1 was used for the activity assay. NC, negative control; Kan, kanamycin (50 μg/mL) as a positive control; DZI, diameter of zone of inhibition (in mm). Data shown representative of three independent experiments (n = 3). Source data are provided as a Source Data file.
