Expression, purification and antimicrobial activity of puroindoline A protein and its mutants
Expression, purification and antimicrobial activity of puroindoline A protein and its mutants
复制标题
DOI：
10.1007/s00726-012-1250-x
复制
发表时间：
2012-03
期刊：
影响因子：
3.5
通讯作者：
Yingjie Miao;Ling Chen;Cheng Wang;Yajuan Wang;Qian Zheng;Chunbao Gao;Guangxiao Yang;G. He
中科院分区：
文献类型：
--
作者：
Yingjie Miao;Ling Chen;Cheng Wang;Yajuan Wang;Qian Zheng;Chunbao Gao;Guangxiao Yang;G. He
小麦嘌呤吲哚啉蛋白质PINA和PINB在决定小麦籽粒硬度以及防御植物抵抗病原体方面起关键作用。PINA具有更强的膜结合特性和抗菌活性，因为它在独特的富含色氨酸的结构域（TRD）中含有更多的色氨酸残基。为了获得抗菌活性更高的蛋白，构建了含有两个或三个TRD拷贝的PINA突变体ABBC和ABBBC，并在E. coliRosetta-gami（DE3）.金属亲和层析用于纯化可溶性亲和标记的重组蛋白。通过在线程序Protein Homology/analog Y Recognition Engine v2.0预测重组蛋白的二级结构，并使用圆二色谱进行实验评估。采用最低抑菌浓度试验和荧光显微镜分析对突变体的抑菌活性进行了评价。结果表明，纯化的重组ABBC正确折叠，对大肠杆菌E. coliandS.金黄色野生型PINA，表明其作为抗微生物剂的潜在用途。结果还证实了TRD是PINA抗菌活性的决定因素，并证明通过添加一个TRD拷贝来增强PINA的抗菌活性是可行的。
Wheat puroindoline proteins, PINA and PINB, play key roles in determining wheat grain hardness as well as in defending the plant against pathogens. PINA has much greater membrane-binding property and antimicrobial activity because it contains more tryptophan residues in the unique tryptophan-rich domain (TRD). In order to obtain proteins with higher antimicrobial activity, mutants of PINA containing two or three copies of TRD, designated ABBC and ABBBC, respectively, were constructed and expressed inE. coliRosetta-gami (DE3). Metal affinity chromatography was used to purify the soluble affinity-tagged recombinant proteins. The secondary structures of the recombinant proteins were predicted by the online program Protein Homology/analog Y Recognition Engine v2.0 and experimentally assessed using circular dichroism. Minimum inhibition concentration tests and fluorescence microscope analyses were employed to evaluate the antimicrobial activities of the mutants. The results showed that the purified recombinant ABBC was correctly folded and presented significantly higher antimicrobial activities againstE. coliandS. aureusthan wild-type PINA, suggesting its potential use as an antimicrobial agent. The results also confirmed that TRD is a determinant of the antimicrobial activity of PINA and demonstrated that it is feasible to enhance the antimicrobial activity of PINA by adding one copy of TRD.