Dos Santos Izabela S, Carvalho André de O, de Souza-Filho Gonçalo A, do Nascimento Viviane V, Machado Olga L T, Gomes Valdirene M
Universidade Estadual do Norte Fluminense, Laboratório de Fisiologia e Bioquímica de Microrganismos, Campos dos Goytacazes 28013-602, RJ, Brazil.
Protein Expr Purif. 2010 May;71(1):8-15. doi: 10.1016/j.pep.2009.11.008. Epub 2009 Dec 3.
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.
植物防御素构成了一类阳离子抗菌肽家族，其具有由四个二硫键稳定的特征性三维折叠模式。这项工作的目的是从豇豆种子中纯化和功能性表达一种防御素，并评估其α-淀粉酶抑制活性。将编码豇豆防御素的cDNA克隆到pET-32 EK/LIC载体中，并用所得构建体转化大肠杆菌细胞。重组肽通过在镍琼脂糖柱上的亲和层析以及使用高效液相色谱在C2/C18柱上的反相层析进行纯化。N端氨基酸测序表明，重组肽的序列与从种子中分离的防御素的序列相似。对天然和重组防御素进行了α-淀粉酶抑制试验。发现豇豆种子防御素可抑制黄斑豆象和赤拟谷盗的α-淀粉酶。α-淀粉酶抑制试验还表明，重组防御素可抑制豆象黄斑豆象的α-淀粉酶。豇豆种子防御素及其重组形式均无法抑制哺乳动物的α-淀粉酶。对重组防御素的三维结构进行了建模，发现所得结构与其他植物防御素的结构相似。
