Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect alpha-amylase inhibitory activity.
Protein Expr Purif
; 71(1): 8-15, 2010 May.
Article
em En
| MEDLINE
| ID: mdl-19948221
Plant defensins make up a family of cationic antimicrobial peptides with a characteristic three-dimensional folding pattern stabilized by four disulfide bridges. The aim of this work was the purification and functional expression of a defensin from cowpea seeds and the assessment of its alpha-amylase inhibitory activity. The cDNA encoding the cowpea defensin was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform Escherichia coli cells. The recombinant peptide was purified via affinity chromatography on a Ni Sepharose column and by reverse-phase chromatography on a C2/C18 column using HPLC. N-terminal amino acid sequencing revealed that the recombinant peptide had a similar sequence to that of the defensin isolated from seeds. The natural and recombinant defensins were submitted to the alpha-amylase inhibition assay. The cowpea seed defensin was found to inhibit alpha-amylases from the weevils Callosobruchus maculatus and Zabrotes subfasciatus. alpha-Amylase inhibition assays also showed that the recombinant defensin inhibited alpha-amylase from the weevil C. maculatus. The cowpea seed defensin and its recombinant form were unable to inhibit mammalian alpha-amylases. The three-dimensional structure of the recombinant defensin was modeled, and the resulting structure was found to be similar to those of other plant defensins.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sementes
/
Bioquímica
/
Defensinas
/
Gorgulhos
/
Escherichia coli
/
Alfa-Amilases
/
Fabaceae
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Estados Unidos