Microbial Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 11 No. 2, Issue of April 15, 2008
© 2008 by Pontificia Universidad Católica de Valparaíso -- Chile Received July 27, 2007 / Accepted January 9, 2008
DOI: 10.2225/vol11-issue2-fulltext-4
RESEARCH ARTICLE

Design and expression of a retro doublet of cecropin with enhanced activity 

Mauricio Díaz
Laboratorio de Genética e Inmunología Molecular
Instituto de Biología
Pontificia Universidad Católica de Valparaíso
Av. Brasil 2950, Valparaíso, Chile
Tel: 56 32 273119
Fax: 56 32 596703
E-mail: mauricio_diazruiz@yahoo.com 

Gloria Arenas
Laboratorio de Genética e Inmunología Molecular
Instituto de Biología
Pontificia Universidad Católica de Valparaíso
Av. Brasil 2950, Valparaíso, Chile
Tel: 56 32 273117
Fax: 56 32 596703
E-mail: garenas@ucv.cl 

Sergio H. Marshall*
Laboratorio de Genética e Inmunología Molecular
Instituto de Biología
Pontificia Universidad Católica de Valparaíso
Av. Brasil 2950, Valparaíso, Chile
Tel: 56 32 273119
Fax: 56 32 596703
E-mail: smarshal@ucv.cl

*Corresponding author

Financial support: This work was supported by the Copec-UC foundation and MECESUP.

Keywords: antimicrobial peptides, Escherichia coli, expression.

Abbreviations:

ACN: acetonitrile
AMP: antimicrobial peptides
CECdir: direct or standard cecropin
CECret: retro cecropin
drAMPs: doublet antimicrobial peptides
IPTG: isopropil-β-D-thio-galactopyranoside
LB: Luria-Bertoni
PCR: polymerase chain reaction
RP-HPLC: reverse phase high performance liquid chromatography

Abstract   Full Text

Novel doublet molecules of cecropin A from Drosophila melanogaster were designed and constructed combining the regular (CECdir) with the inverted (CECret) coding sequence of the standard CEC A1 gene resulting in the following configurations: CECdir-CECretand CECret-CECdir. These two recombinant molecules were generated using a three-primer driven PCR reaction yielding composite single functional aminoacidic molecules with the coding sequences of CECdir linked in frame with the coding sequence of CECret and vice versa. In order to obtain these constructions, a retropeptide DNA-coding sequence was chemically synthesized to match the expected polarity of the newly generated CECret sequence. Both doublet antimicrobial peptides (drAMPs) were cloned in the T7 promoter driven expression plasmid pET27b+ and expressed in E. coli BL21 without any fusion protein. Only the former recombinant peptide was expressed and purified from cell extracts and its specific activity against two different bacteria showed to be higher than those displayed by their monomer parental counterparts.

Supported by UNESCO / MIRCEN network