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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 15, No. 2, 2012, pp. 1-10
Bioline Code: ej12015
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 15, No. 2, 2012, pp. 1-10

 en Intein-mediated expression of cecropin in Escherichia coli check for this species in other resources
Díaz, Mauricio; Venturini, Elena; Marchetti, Stefano; Arenas, Gloria & Marshall, Sergio H.

Abstract

Abstract Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli check for this species in other resources ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii check for this species in other resources , Vibrio alginolyticus check for this species in other resources and Escherichia coli.

Keywords
antimicrobial; cecropin; fusion; intein; peptide; soluble

 
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