酵母组蛋白乙酰转移酶Elp3多克隆抗体的制备、鉴定及应用

Identification and application of yeast histone acetyltransferases Elp3 polyclonal antibody

摘要:

酵母Elp3(Yeast elongation protein 3, yElp3)是组蛋白乙酰转移酶复合物Elongator的催化亚基, 通过组蛋白乙酰化参与基因转录延伸。近来证实胞质中也存在的Elp3还参与胞外分泌、tRNA修饰等重要生物学过程。为深入研究Elp3的复杂功能, PCR法克隆pYES2-yElp3质粒中编码yElp3N末端亲水区段(22~94aa), 构建原核表达载体pMXB10-yElp3- 219, 经IPTG诱导表达和几丁质柱纯化后免疫兔制备多克隆抗体。ELISA检测显示该抗体有较高的效价(不低于1:2500), 免疫印迹结果表明该抗体可特异性识别表达纯化及酵母细胞中的Elp3蛋白, 运用该抗体进行的染色质免疫沉淀揭示转入elp3?菌株的酵母/人融合的yhElp3之所以可补偿突变株的基因表达延迟缺陷, 是因其直接参与SSA3基因的转录延伸。

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 elp3? cells.