IN-VIVO MONITORING-SYSTEM FOR STRUCTURE-FUNCTION RELATIONSHIP ANALYSIS OF THE ANTIBACTERIAL PEPTIDE APIDAECIN

A unique antibacterial peptide derivative found in immune honeybee lym ph, apidaecin 1b (AP1), was randomly mutagenized and characterized by a newly established system to analyze in vivo its structure-function r elationship. 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) molecul e. Expression of the SSI-AP1 fusion protein was found to depend on the concentration of the transcriptional inducer isopropyl-beta-D-thio-ga lactopyranoside (IPTG) and to parallel the degree of growth inhibition of the transformant cells. Subsequently, apidaecin derivatives produc ed by localized random mutagenesis were screened with this IPTG concen tration-controlled in vivo system by monitoring the growth inhibition patterns of the transformant cells. One mutant apidaecin (P9L) that ha d reduced activity was purified and isolated from the periplasmic frac tion of an L. coli transformant. Its antibacterial activity was reduce d to one-third of that of wild-type apidaecin. When considered togethe r 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.