Intein-mediated expression of cecropin in Escherichia coli



Título del documento: Intein-mediated expression of cecropin in Escherichia coli
Revista: Electronic journal of biotechnology
Base de datos: PERIÓDICA
Número de sistema: 000358458
ISSN: 0717-3458
Autores: 1
2
2
3
3
Instituciones: 1Pontificia Universidad Católica de Valparaíso, Escuela de Ingeniería Bioquímica, Valparaíso. Chile
2Universita di Udine, Dipartimento di Scienze Agrarie e Ambientali, Udine, Friuli Venezia Giuli. Italia
3Pontificia Universidad Católica de Valparaíso, Facultad de Ciencias, Valparaíso. Chile
Año:
Volumen: 15
Número: 2
País: Chile
Idioma: Inglés
Tipo de documento: Artículo
Enfoque: Experimental, aplicado
Resumen en inglés 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 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, Vibrio alginolyticus and Escherichia coli
Disciplinas: Biología
Palabras clave: Biotecnología,
Bioquímica,
Péptidos antimicrobianos,
Cecropinas,
Expresión génica,
Fusión celular,
Escherichia coli
Keyword: Biology,
Biotechnology,
Biochemistry,
Antimicrobial peptides,
Cecropins,
Gene expression,
Cell fusion,
Escherichia coli
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