Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect α-amylase inhibitory activity

• Published in: Protein expression and purification Vol. 71; no. 1; pp. 8 - 15
• Main Authors: dos Santos, Izabela S., Carvalho, André de O., de Souza-Filho, Gonçalo A., do Nascimento, Viviane V., Machado, Olga L.T., Gomes, Valdirene M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-05-2010
• Subjects: alpha-Amylases - antagonists & inhibitors; Amino Acid Sequence; Animals; Biochemistry - methods; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Cowpea seeds; Defensins - chemistry; Defensins - isolation & purification; Defensins - pharmacology; Electrophoresis, Polyacrylamide Gel; Enzyme Assays; Escherichia coli - drug effects; Escherichia coli - metabolism; Fabaceae - chemistry; Fabaceae - drug effects; Functional expression; Models, Molecular; Molecular Sequence Data; Plant defensin; Seeds - chemistry; Sequence Analysis, Protein; Weevils - drug effects; Weevils - enzymology; α-Amylase inhibitor; Plant defensin; Functional expression; Cowpea seeds; α-Amylase inhibitor
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2009.11.008

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 α-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 α-amylase inhibition assay. The cowpea seed defensin was found to inhibit α-amylases from the weevils Callosobruchus maculatus and Zabrotes subfasciatus. α-Amylase inhibition assays also showed that the recombinant defensin inhibited α-amylase from the weevil C. maculatus. The cowpea seed defensin and its recombinant form were unable to inhibit mammalian α-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.