Bactericidal action of lysozymes attached with various sizes of hydrophobic peptides to the C-terminal using genetic modification.
The genetic modification of lysozyme was attempted to improve the bactericidal activity against Gram-negative bacteria E. coli. The different lengths of hydrophobic peptides were attached to the C-terminus of the hen egg white lysozyme to investigate the most effective length of the hydrophobic peptides for killing bacteria. The oligonucleotides encoding Phe-Val-Pro (H3), Phe-Phe-Val-Ala-Pro (H5) and Phe-Phe-Val-Ala-Ile-Ile-Pro (H7) were fused to the C-terminus Leu 129 of lysozyme cDNA. The reconstructed cDNAs were inserted into the yeast expression vector. The hydrophobic peptide-fused lysozymes were secreted in the yeast carrying the reconstructed cDNA. Although the hydrophobic peptide-fused lysozymes retained 75 80% lytic activity of the wild-type protein, the bactericidal action to E. coli was greatly increased with the length of hydrophobic peptides. These results suggest that the hydrophobic peptides play an important role in killing Gram-negative bacteria. To elucidate the role of catalytic domain in bactericidal action of the hydrophobic fusion lysozyme (H5-Lz), the mutant hydrophobic lysozyme (H5/E35A-Lz) whose glutamic acid was substituted with alanine at the position 35 was constructed to diminish the catalytic activity. The mutant hydrophobic lysozyme (H5/E35A-Lz) was greatly lost the bactericidal action to E. coli, suggesting that not only the length of hydrophobic peptide fused to C-terminus but also the catalytic domain is important for the bactericidal action of the hydrophobic peptide-fused lysozyme.
Arima
H
H
Department of Biological Chemistry, Yamaguchi University, Japan.
Ibrahim
H R
HR
Kinoshita
T
T
Kato
A
A
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents / Q000737:chemistry / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development; D015870:Gene Expression; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D000911:Antibodies, Monoclonal; D008070:Lipopolysaccharides; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D009113:Muramidase