Wang Xiaoyu, Li Ming, Zhang Jiahuan, Zhang Yan, Zhang Guiying, Wang Jinsheng
Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China.
Mol Biol Rep. 2007 Sep;34(3):189-98. doi: 10.1007/s11033-006-9034-6. Epub 2006 Dec 19.
In the current study, we identified a key functional region in harpins from Xanthomonas that suppressed protein aggregation and mediated its expression in E. coli. Our data suggested that the presence of two common features in harpins [Wei et al. (1992) Science 257:85-88], namely, high glycine content and lack of cysteine residues, were not sufficient for Xanthomonas to elicit hypersensitive response (HR) activity or heat stability. Additionally, bioinformatic analyses revealed that the secondary structure of a conserved N-terminal region consisting of 12 highly hydrophilic amino acids (QGISEKQLDQLL) was alpha-helical. Following site-directed mutagenesis deletion of this region, the three mutated harpin proteins, in cultures induced at 37 degrees C, failed to elicit a HR in tobacco leaves. However, at 24 degrees C, two mutated harpins retained the ability to elicit HR, albeit with lower expression levels than that noted with the wild-type. SDS-PAGE and Western blot data suggested the HpaG mutant protein was found almost entirely in the inclusion body. These data demonstrated that these conserved amino acid residues played a critical role in protein aggregation and inclusion body formation in harpins from Xanthomonas.
在本研究中，我们在来自黄单胞菌的harpin蛋白中鉴定出一个关键功能区域，该区域可抑制蛋白质聚集并介导其在大肠杆菌中的表达。我们的数据表明，harpin蛋白中的两个共同特征[Wei等人（1992年）《科学》257:85 - 88]，即高甘氨酸含量和缺乏半胱氨酸残基，不足以使黄单胞菌引发过敏反应（HR）活性或热稳定性。此外，生物信息学分析表明，由12个高度亲水氨基酸（QGISEKQLDQLL）组成的保守N端区域的二级结构为α螺旋。对该区域进行定点诱变缺失后，在37℃诱导培养的三种突变harpin蛋白未能在烟草叶片中引发HR。然而，在24℃时，两种突变harpin蛋白仍保留引发HR的能力，尽管其表达水平低于野生型。SDS - PAGE和蛋白质免疫印迹数据表明，HpaG突变蛋白几乎完全存在于包涵体中。这些数据证明，这些保守氨基酸残基在来自黄单胞菌的harpin蛋白的蛋白质聚集和包涵体形成中起关键作用。
