Liu Yun, Zheng Yizhi
Institute of Genetics and Cytology, School of Life Science, Northeast Normal University, Changchun 130024, PR China.
Biochem Biophys Res Commun. 2005 May 27;331(1):325-32. doi: 10.1016/j.bbrc.2005.03.165.
To have knowledge of the effect of soybean PM2 protein in protecting dehydrated cells and its functional region, PM2 cDNA was isolated from soybean immature seeds. The recombinants expressing full-length PM2, truncated polypeptides of PM2A (aa 1-262) or PM2B (aa 129-262, 22-mer repeating region), or artificial polypeptide PM2C (duplication of 22-mer repeating region) were constructed. By using SDS-PAGE and mass spectrometry approaches, these fusion polypeptides were identified and proved to be hydrophilic and heat-stable. Spot assays of BL/PM2 and BL/pET28 (as control) showed that protein PM2 increased salt tolerance (500 mM NaCl or 500 mM KCl) of Escherichia coli, rather than osmotic tolerance (1100 mM sorbitol). In addition, comparing the survival ratios of the transformants under 500 mM NaCl or 500 mM KCl stresses, the results showed that: (1) the survival ratios of BL/PM2 and BL/PM2B were quite similar, both showing much higher values than those of BL/pET28. (2) The survival ratios of BL/PM2C were much higher than those of BL/PM2, BL/PM2A, and BL/PM2B. This provides the first experimental evidence that PM2 polypeptide enhances salt tolerance of E. coli cells, and the 22-mer repeat region is an important functional region.
为了解大豆PM2蛋白在保护脱水细胞中的作用及其功能区域，从大豆未成熟种子中分离出PM2 cDNA。构建了表达全长PM2、PM2A截短多肽（氨基酸1 - 262）或PM2B截短多肽（氨基酸129 - 262，22聚体重复区域）或人工多肽PM2C（22聚体重复区域重复）的重组体。通过SDS - PAGE和质谱方法，鉴定了这些融合多肽，并证明它们具有亲水性和热稳定性。BL/PM2和BL/pET28（作为对照）的斑点试验表明，PM2蛋白提高了大肠杆菌的耐盐性（500 mM NaCl或500 mM KCl），而不是渗透压耐受性（1100 mM山梨醇）。此外，比较500 mM NaCl或500 mM KCl胁迫下转化体的存活率，结果表明：（1）BL/PM2和BL/PM2B的存活率非常相似，均远高于BL/pET28。（2）BL/PM2C的存活率远高于BL/PM2、BL/PM2A和BL/PM2B。这提供了首个实验证据，即PM2多肽增强了大肠杆菌细胞的耐盐性，且22聚体重复区域是一个重要的功能区域。
