In vitro reconstitution of antimicrobial pathogen activity by expressed recombinant bovine lactoferrin N-terminal peptide in Escherichia coli.
Recombinant bovine lactoferrin N-terminal polypeptide (rbLF-N) Escherichia coli expression system was constructed and the rbLF-N antimicrobial activity was displayed by enzymatic proteolysis in this study. A 162 bp 5'-terminal fragment of bovine lactoferrin (bLF) gene from bovine liver gDNA was amplified by PCR. The DNA fragment containing exon-2 of the bLF gene was cloned into the expression vector pGEX-4T1 and the glutathione-S-transferase-rbLF-N (GST-rbLF-N) fusion protein was obtained by over-expression in Esch. coli BL21(DE3). After thrombin/pepsin digestion, the rbLF-N was released from the fusion protein. The recombinant peptide was separated and identified by SDS-PAGE, HPLC and LC-MS/MS analysis. A very strong anti-food-born microbial pathogen activity of the rbLF-N peptides was displayed through bio- and kinetic-assays in vitro. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the rbLF-N peptide for bacterial pathogens Staphylococcus aureus, Streptococcus mutans, Esch. coli and Klebsiella pneumoniae were 11.7, 11.7, 11.7, 23.4 microg and 23.4, 11.7, 11.7, 46.4 microg, respectively. This study created a new route for exploring lactoferrin peptide application in food science.
Luo
Hongxia
H
Key Lab of Functional Dairy Science of Beijing and Ministry of Education of China, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China 100083.
Chen
Shangwu
S
Ren
Fazheng
F
Guo
Huiyuan
H
Lin
Shaohua
S
Xu
Wentao
W
D000818:Animals; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000627:therapeutic use; D001483:Base Sequence; D002415:Cats; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005784:Gene Amplification; D007711:Klebsiella pneumoniae / Q000187:drug effects; D007781:Lactoferrin / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000627:therapeutic use*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000627:therapeutic use; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000187:drug effects
D000900:Anti-Bacterial Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007781:Lactoferrin