Purification of novel peptide antibiotics from human milk.
A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-K (residues 63-117) and inhibits the growth of gram-positive, gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.
Lower Saxony Institute for Peptide Research (IPF), Hannover, Germany. cliepke@gmx.de
C
C
Liepke
H D
HD
Zucht
W G
WG
Forssmann
L
L
Ständker
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D002364:Caseins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000900:Anti-Bacterial Agents; D002364:Caseins; D010446:Peptide Fragments