S. Taguchi et al., IN-VIVO MONITORING-SYSTEM FOR STRUCTURE-FUNCTION RELATIONSHIP ANALYSIS OF THE ANTIBACTERIAL PEPTIDE APIDAECIN, Applied and environmental microbiology, 60(10), 1994, pp. 3566-3572
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.