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Author (up) Dos Santos, I.S.; Carvalho, A. de O.; de Souza-Filho, G.A.; do Nascimento, V.V.; Machado, O.L.T.; Gomes, V.M. url  doi
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  Title Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect alpha-amylase inhibitory activity Type Journal Article
  Year 2010 Publication Protein Expression and Purification Abbreviated Journal Protein Expr Purif  
  Volume 71 Issue 1 Pages 8-15  
  Keywords Amino Acid Sequence; Animals; Biochemistry/*methods; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Defensins/chemistry/*isolation & purification/pharmacology; Electrophoresis, Polyacrylamide Gel; Enzyme Assays; Escherichia coli/drug effects/*metabolism; Fabaceae/*chemistry/drug effects; Models, Molecular; Molecular Sequence Data; Seeds/*chemistry; Sequence Analysis, Protein; Weevils/drug effects/*enzymology; alpha-Amylases/*antagonists & inhibitors  
  Abstract 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.  
  Address Universidade Estadual do Norte Fluminense, Laboratorio de Fisiologia e Bioquimica de Microrganismos, Campos dos Goytacazes 28013-602, RJ, Brazil  
  Corporate Author Thesis  
  Publisher Place of Publication Editor  
  Language English Summary Language Original Title  
  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 1046-5928 ISBN Medium  
  Area Expedition Conference  
  Notes PMID:19948221 Approved no  
  Call Number WNEC @ jrocheleau @ Serial 31  
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