Production of an Arabidopsis halleri foliar defensin in Escherichia coli.
J Appl Microbiol
; 106(5): 1640-8, 2009 May.
Article
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| MEDLINE
| ID: mdl-19226399
ABSTRACT
AIMS:
Production of the recombinant Arabidopsis halleri defensin AhPDF1.1 in a native-like form. METHODS ANDRESULTS:
Mature AhPDF1.1 cDNA was cloned into pET-28-a(+) and expressed in Escherichia coli Rosetta. After a denaturing extraction, purification by metal affinity chromatography and CNBr cleavage of the His-tag, a protein without extra amino acids at the N-terminus was obtained. An oxidative folding step was then required to renature the protein that was then purified to homogeneity by a C18 HPLC separation. Mass spectroscopy and circular dichroism analyses showed that the recombinant AhPDF1.1 has the expected molecular mass and 3D-structure features of a folded defensin with four-disulfide bridges. The recombinant protein is active against the filamentous fungus Fusarium oxysporum with a minimal inhibitory concentration of 0.6 micromol l(-1).CONCLUSION:
The proposed purification protocol produces a native-like defensin suitable for tests of new biological roles. SIGNIFICANCE AND IMPACT OF THE STUDY Plant defensins are essentially known as anti-fungal proteins; however, some unexpected actions on plant cells have recently been discovered. AhPDF1.1, for example, has been shown to confer zinc tolerance. Efficient production of native-like defensins is required to explore the different targets and roles of plant defensins.
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Base de datos:
MEDLINE
Asunto principal:
Proteínas de Plantas
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Arabidopsis
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Hojas de la Planta
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Defensinas
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Escherichia coli
Idioma:
En
Revista:
J Appl Microbiol
Asunto de la revista:
MICROBIOLOGIA
Año:
2009
Tipo del documento:
Article