In vivo monitoring system for structure-function relationship analysis of the antibacterial peptide apidaecin.
A unique antibacterial peptide derivative found in immune honeybee lymph, apidaecin 1b (AP1), was randomly mutagenized and characterized by a newly established system to analyze in vivo its structure-function relationship. Initially, a high-level expression host-vector system for AP1 in Escherichia coli was constructed by creating a fusion protein with the highly stable Streptomyces subtilisin inhibitor (SSI) molecule. Expression of the SSI-AP1 fusion protein was found to depend on the concentration of the transcriptional inducer isopropyl-beta-D-thio-galactopyranoside (IPTG) and to parallel the degree of growth inhibition of the transformant cells. Subsequently, apidaecin derivatives produced by localized random mutagenesis were screened with this IPTG concentration-controlled in vivo system by monitoring the growth inhibition patterns of the transformant cells. One mutant apidaecin (P9L) that had reduced activity was purified and isolated from the periplasmic fraction of an E. coli transformant. Its antibacterial activity was reduced to one-third of that of wild-type apidaecin. When considered together with the other mutations, it was concluded that several Pro residues, including that at the ninth position, are responsible for expression of the antibacterial action of apidaecin.
Taguchi
S
S
Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.
Nakagawa
K
K
Maeno
M
M
Momose
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides; D001483:Base Sequence; D001516:Bees / Q000235:genetics; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017344:Genes, Insect; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D017931:DNA Primers; D010455:Peptides; D011993:Recombinant Fusion Proteins; C061361:apidaecin; D004247:DNA