bLF (20% iron saturated) was kindly provided by DMV International (Veghel, The Netherlands). LF derived peptides (Table 1) were synthesized by solid phase peptide synthesis using Fmoc chemistry with a Siro II synthesizer (Biotage, Uppsala, Sweden) according to the manufacturer’s protocol. Purification by Reverse Phase-HPLC was conducted as described previously (Bolscher et al. 2009 (link)). Identity of the peptides was confirmed by mass spectrometry (Bruker Daltonik GMBH, Bremen, Germany) and molar concentrations were calculated based on their weight.
Sequences and characteristics of the peptides investigated
Peptidea
Primary structure
Charge
MBC (μM)b
Y. e.
Y. p.
LFampin265–284
DLIWKLLSKAQEKFGKNKSR
+ 4
6.3
ND
LFcin17–30
FKCRRWQWRMKKLG
+ 6
0.8
ND
LFchimerac
+ 12
0.2
1.6
LFampin265–284 and LFcin17–30
DLIWKLLSKAQEKFGKNKSRand FKCRRWQWRMKKLG
+ 10
0.8
ND
Y. e.: Y. enterocolitica, Y. p.: Y. pseudotuberculosis
aThe purity of the peptides was at least 95% and the authenticity of the peptides was confirmed by MALDI-TOF mass spectrometry
bMinimal bactericidal concentration (MBC), in 1 mM phosphate buffer (from Sijbrandij et al. 2017 (link)). ND: not detected up to 50 µM peptide
cA single C-terminal lysine amide (K) was substituted at the α- and ε-amino groups with the two peptides via the C-terminal site, while leaving the two N-termini as free ends
Sijbrandij T., Ligtenberg A.J., Nazmi K., van den Keijbus P.A., Veerman E.C., Bolscher J.G, & Bikker F.J. (2018). LFchimera protects HeLa cells from invasion by Yersinia spp. in vitro. Biometals, 31(6), 941-950.
Minimal bactericidal concentration (MBC) against Y. enterocolitica
Minimal bactericidal concentration (MBC) against Y. pseudotuberculosis
control variables
1 mM phosphate buffer
controls
Positive control: Not specified
Negative control: Not specified
Annotations
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