Functional expression and activity of the recombinant antifungal defensin PvD1r from Phaseolus vulgaris L. (common bean) seeds.
Functional expression and activity of the recombinant antifungal defensin PvD1r from Phaseolus vulgaris L. (common bean) seeds.
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DOI：
10.1186/1471-2091-15-7
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发表时间：
2014-04-01
期刊：
影响因子：
--
通讯作者：
Gomes VM
中科院分区：
文献类型：
--
作者：
Mello Ede O;dos Santos IS;Carvalho Ade O;de Souza LS;de Souza-Filho GA;do Nascimento VV;Machado OL;Zottich U;Gomes VM
防御素是基本的，富含半胱氨酸的抗菌肽，是植物防御病原体的重要组成部分。此前，我们从菜豆种子中分离到了一种防御素PvD1。本研究的目的是在原核系统中过表达PvD1，通过比较PvD1r与天然防御素PvD1的抑菌活性来验证重组PvD1 （PvD1r）的生物学功能，并利用缺乏鞘脂生物合成基因的白色念珠菌突变株来揭示PvD1r在白色念珠菌细胞中的靶位。将先前获得的编码PvD1的cDNA克隆到pET-32 EK/LIC载体中，并利用所构建的构建体转化细菌细胞（Rosetta Gami 2 (DE3) pLysS）导致重组蛋白的表达。诱导表达后，纯化PvD1r，用肠激酶裂解，再用层析步骤纯化。n端氨基酸测序结果表明，重组PvD1r的整个过程，包括与肠激酶的裂解，都是成功的。此外，建模显示PvD1r具有与从植物中分离的防御素相似的结构。纯化的PvD1和PvD1r对野生型病原菌白色念珠菌的生长具有抑制作用。然而，这两种防御素对白色念珠菌突变株没有表现出抑制活性。对野生型白色念珠菌进行抗真菌实验后，光镜下观察其形态变化。PvD1r与FITC偶联，随后用DAPI处理野生型白色念珠菌，发现标记的肽在细胞内定位。在突变株中，未检测到细胞内标记。我们的研究结果表明，PvD1r在重组生产、肠激酶裂解和纯化后保持了充分的生物活性。此外，我们的抗菌实验、显微分析和PvD1r-FITC标记实验的结果相互证实，并使我们认为PvD1在白色念珠菌细胞中的靶点是鞘脂糖基神经酰胺。
Defensins are basic, cysteine-rich antimicrobial peptides that are important components of plant defense against pathogens. Previously, we isolated a defensin, PvD1, from Phaseolus vulgaris L. (common bean) seeds. The aim of this study was to overexpress PvD1 in a prokaryotic system, verify the biologic function of recombinant PvD1 (PvD1r) by comparing the antimicrobial activity of PvD1r to that of the natural defensin, PvD1, and use a mutant Candida albicans strain that lacks the gene for sphingolipid biosynthesis to unravel the target site of the PvD1r in C. albicans cells. The cDNA encoding PvD1, which was previously obtained, was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform bacterial cells (Rosetta Gami 2 (DE3) pLysS) leading to recombinant protein expression. After expression had been induced, PvD1r was purified, cleaved with enterokinase and repurified by chromatographic steps. N-terminal amino acid sequencing showed that the overall process of the recombinant production of PvD1r, including cleavage with the enterokinase, was successful. Additionally, modeling revealed that PvD1r had a structure that was similar to the defensin isolated from plants. Purified PvD1 and PvD1r possessed inhibitory activity against the growth of the wild-type pathogenic yeast strain C. albicans. Both defensins, however, did not present inhibitory activity against the mutant strain of C. albicans. Antifungal assays with the wild-type C. albicans strains showed morphological changes upon observation by light microscopy following growth assays. PvD1r was coupled to FITC, and the subsequent treatment of wild type C. albicans with DAPI revealed that the labeled peptide was intracellularly localized. In the mutant strain, no intracellular labeling was detected. Our results indicate that PvD1r retains full biological activity after recombinant production, enterokinase cleavage and purification. Additionally, our results from the antimicrobial assay, the microscopic analysis and the PvD1r-FITC labeling assays corroborate each other and lead us to suggest that the target of PvD1 in C. albicans cells is the sphingolipid glucosylceramide.
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影响因子：
2.9
作者：
Lobo, Denise S.;Pereira, Iuri B.;Kurtenbach, Eleonora
通讯作者：
Kurtenbach, Eleonora
DOI：
10.1111/j.1432-1033.1991.tb15780.x
发表时间：
1991-02-26
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
BONTEMS, F;ROUMESTAND, C;TOMA, F
通讯作者：
TOMA, F
DOI：
10.1016/j.bbrc.2004.02.173
发表时间：
2004-04-16
影响因子：
3.1
作者：
Elmorjani, K;Lurquin, V;Marion, D
通讯作者：
Marion, D
影响因子：
3.5
作者：
INOUE, H;NOJIMA, H;OKAYAMA, H
通讯作者：
OKAYAMA, H
影响因子：
5.8
作者：
Dorcinvil, Ronald;Sotomayor-Ramirez, David;Beaver, James
通讯作者：
Beaver, James