八肋游仆虫酸性核糖体磷酸化蛋白P1有2个亚型

Ciliates Euplotes octocarinatus Expresses Two Different Sub-forms of Acidic Ribosomal Protein P1

真核生物酸性核糖体磷酸化蛋白(P0、P1、P2)位于核糖体60S大亚基上,它们在核糖体上共同组成一个向外侧凸出的五聚体的柄状复合物[P0·(P1·P2)2],该复合物在蛋白质合成延伸过程中起着重要作用.为了探讨单细胞真核生物核糖体柄状复合物的组成形式及在蛋白质合成中的作用,对八肋游仆虫(Euplotes octocarinatus)的P1进行了研究.通过生物信息学方法,分析八肋游仆虫基因组及转录组数据,找到2个酸性核糖体蛋白P1基因,从DNA和c DNA中都扩增到这2个P1基因,表明八肋游仆虫酸性核糖体磷酸化蛋白P1确实存在2个亚型.将2个基因克隆后分别构建重组表达质粒p ET28a-P1A和p GEX-6P-1-P1B,在大肠杆菌BL21中获得高效表达.经镍柱和GST柱亲和层析后,获得较高纯度的八肋游仆虫酸性核糖体蛋白Eo P1A和Eo P1B,表达产物经Western印迹检测为阳性.Pull-down分析了Eo P1A和Eo P1B之间的相互作用.结果表明,游仆虫酸性核糖体磷酸化蛋白P1的2个亚型Eo P1A和Eo P1B之间存在相互作用.

Acidic ribosomal phosphoproteins（ P0,P1,P2） resided on the large subunit of eukaryotic60 S ribosomes constitute a lateral protuberance ribosomal stalk complex. The complex was indicated to play an important role during the elongation stage of protein biosynthesis. To understand the assembling and function of ribosomal stalk in lower eukaryotic organisms and the involved mechanisms, E.octocarinatus ribosomal protein P1 was used in this study. Two P1 genes were identified from the transcriptome and the genome of E. octocarinatus. Two recombinant plasmids（ p ET28a-P1 A and p GEX-6P-1-P1B） were constructed and used to transform E. coli BL21 for expression. Recombinant proteins of Eo P1 A and Eo P1 B were purified by Ni-NTA or GST affinity chromotography. The purified proteins were analyzed by Western blot for purity and yields. The interaction between Eo P1 A and Eo P1 B was tested by pull-down assay. Our results indicated that two forms of acidic ribosomal phosphoprotein P1（ Eo P1 A and Eo P1B） were existed in E. octocarinatus,which were able to interact with each other.