Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system

Previously, we established for the first time an in vivo monitoring assay s ystem conjugated with random mutagenesis in order to study the structure-fu nction relationship of the antimicrobial peptide, apidaecin [Taguchi ct al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, thi s methodology was used to carry out the functional mapping of a second targ et, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum ct al. (1 996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene enc oding thanatin was expressed in a fused form with Streptomyces protease inh ibitor protein, SSI, under the control of tac promoter in Escherichia coil JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta -D-thio-galact opyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the stru ctural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony siz e (plate assay) or optical density (liquid assay) in comparison with the wi ld-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by t he in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubl y mutated protein (K17R/R20G), were stably overproduced with a fusion. part ner protein resulting in the efficient formation of inclusion bodies in E. coil BL21(DE3). The products were isolated in large amounts (yield 30%) fro m the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coil JM109 activities, judged by minimu m inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on th e NMR solution structure and molecular dynamics, the structure- function re lationship of thanatin is discussed by comparing the functional mapping dat a obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within t he C-terminal loop joining the beta -strands arranged antiparallel to one a nother that are supposed to be crutial for exhibiting anti-E. coil activity .