Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect alpha-amylase inhibitory activity.
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.
Dos Santos
Izabela S
IS
Universidade Estadual do Norte Fluminense, Laboratório de Fisiologia e Bioquímica de Microrganismos, Campos dos Goytacazes 28013-602, RJ, Brazil.
Carvalho
André de O
Ade O
de Souza-Filho
Gonçalo A
GA
do Nascimento
Viviane V
VV
Machado
Olga L T
OL
Gomes
Valdirene M
VM
D000595:Amino Acid Sequence; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D023082:Defensins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D007887:Fabaceae / Q000737:chemistry* / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012639:Seeds / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D048889:Weevils / Q000187:drug effects / Q000201:enzymology*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D023082:Defensins; D000516:alpha-Amylases