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

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

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

S Taguchi,K Kuwasako,A Suenaga,M Okada,H Momose,

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused 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. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.

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

S Taguchi,K Kuwasako,A Suenaga,M Okada,H Momose,

For similar biochemical phenomena, metabolism, and nutrition: biochemical phenomena: structure-activity relationship research abstracts see: biochemical phenomena, metabolism, and nutrition: biochemical phenomena: structure-activity relationship research

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

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 128

Page Numbers: 745-54

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Nov

YEAR: 2000

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

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LANGUAGE: eng

NlmUniqueID: 376600

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

KEYWORDS: Structure-Activity Relationship

MESH TERMS: pharmacology

Chemical & Substance for Abstract: Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system. Information

Substance Name: thanatin

Registry Number: 0

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

AFFILIATION: Department of Biological Science and Technology, Science University of Tokyo, Yamazaki, Noda, Chiba 278-8510, Japan. staguchi@postman. riken.go.jp

Country: JAPAN

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MEDLINETA: J Biochem

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