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[酵母组蛋白乙酰转移酶Elp3多克隆抗体的鉴定与应用]

[Identification and application of yeast histone acetyltransferases Elp3 polyclonal antibody].

作者信息

Li Fen, Tian Shujuan, Zhang Shuai, Kong Yan, Wang Yanfang

机构信息

College of Life Sciences, Henan Normal University, Xinxiang 453007, China.

出版信息

Sheng Wu Gong Cheng Xue Bao. 2009 Aug;25(8):1261-6.

Abstract

Yeast Elongation protein 3 (yElp3), the catalytic subunit of the multi-subunit histone acetyltransferase elongator complex, is involved in histone acetylation and transcription, exocytosis and tRNA modification. To study the complex function of yElp3 in yeast, we amplified the yElp3 gene fragment encoding 73aa in the N-terminal from plasmid pYES2-yElp3, and then cloned it into pMXB10 to construct the recombinant plasmid pMXB10-yElp3-219. We expressed the fusion protein in E. coli BL21 (DE3), then purified it by chin affinity column, and finally obtained the soluble purified protein (8.0 kD), which was used to immune the rabbits for acquiring antiserum. ELISA and Western blotting indicated that the polyclonal antibody was of high titration and specificity. Chromatin immunoprecipitation (ChIP) assay with this antibody suggested that yhElp3 exerted the transcriptional regulatory function directly through its presence on the SSA3 gene; this might be the reason that it can rescue the delay activation of SSA3 in elp3delta cells.

摘要

酵母延伸蛋白3(yElp3)是多亚基组蛋白乙酰转移酶延伸因子复合物的催化亚基,参与组蛋白乙酰化与转录、胞吐作用及tRNA修饰。为研究yElp3在酵母中的复杂功能,我们从质粒pYES2 - yElp3中扩增出编码N端73个氨基酸的yElp3基因片段,然后将其克隆到pMXB10中构建重组质粒pMXB10 - yElp3 - 219。我们在大肠杆菌BL21(DE3)中表达融合蛋白,然后通过几丁质亲和柱进行纯化,最终获得可溶性纯化蛋白(8.0 kD),该蛋白用于免疫兔子以获取抗血清。ELISA和蛋白质印迹表明多克隆抗体具有高滴度和特异性。用该抗体进行的染色质免疫沉淀(ChIP)分析表明,yhElp3通过其在SSA3基因上的存在直接发挥转录调控功能;这可能是它能够挽救elp3δ细胞中SSA3延迟激活的原因。

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