恶性疟原虫组蛋白甲基转移酶SET7催化结构域的表达及活性鉴定

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摘要目的：构建杆状病毒昆虫表达质粒pFast-N-set7cd、pFast-C-set7cd和无细胞麦胚表达载体pEU-His-set7cd，表达恶性疟原虫组蛋白甲基转移酶SET7催化结构域（PfSET7cd），并鉴定PfSET7cd的组蛋白甲基转移酶活性。方法：通过分子克隆技术构建获得pFast-N/C-set7cd以及pEU-His-set7cd表达质粒，尝试通过杆状病毒传代方式与无细胞麦胚表达方式获得重组PfSET7cd蛋白，以SDS-PAGE和Western blot鉴定表达产物，并进一步检测重组蛋白的酶活性。结果：用于昆虫表达的重组质粒pFast-N-set7cd、pFast-C-set7cd双酶切结果大小与测序结果均正确，但是Western blot并未检测到其在昆虫细胞中的可溶性表达产物；pEU-His-set7cd通过无细胞麦胚表达系统表达获得的蛋白与预测大小一致，经Western blot鉴定正确，并用NTA-Ni2+亲和层析纯化。体外酶活实验显示PfSET7cd具有催化H3第36位赖氨酸上三甲基化（H3K36me3）活性。结论：通过无细胞表达系统获得可溶性的PfSET7cd重组蛋白，PfSET7cd重组蛋白具有介导H3K36me3的活性，但不影响H3K4me3和H3K9me3水平。

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	张亮亮
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	魏启美
	江陆斌
	梁韶晖

关键词 ：恶性疟原虫, 组蛋白甲基转移酶, 重组蛋白表达, 无细胞麦胚表达系统, 活性鉴定

Abstract：Objective: To construct the recombinant baculovirus expression plasmid pFast-N-set7cd, pFast-C-set7cd and cell-free wheat germ expression vector pEU-His-set7cd, to express Plasmodium falciparum histone-lysine N-methyltransferase SET7 catalytic domain (PfSET7cd), and to identify PfSET7cd protein methyltransferase activity. Methods: pFast-N/C-set7cd and pEU-His-set7cd were constructed for expression of recombinant PfSET7cd by baculovirus-insect cell system and wheat germ cell-free expression system, respectively, and recombinant protein was identified by SDS-PAGE and Western blotting. Furthermore, recombinant protein was purified and applied in vitro enzymatic activity assay. Results: The recombinant plasmid pFast-N-set7cd and pFast-C-set7cd were constructed successfully and conformed by sequencing. However, no soluble PfSET7cd protein was detected by Western blotting in insect cells. By wheat germ cell-free expression system, soluble recombinant PfSET7cd was expressed and the molecular weight of protein agreed well with the theoretical prediction, which was identified by Western blot. And then, recombinant PfSET7cd was purified with NTA-Ni2+ affinity column. In vitro enzymatic activity assay showed that PfSET7cd exhibites H3K36me3 activity. Conclusion: Soluble recombinant protein, PfSET7cd, can be successfully obtained by wheat germ cell-free expression system, and this protein can catalyze H3K36 trimethylation (H3K36me3) in vitro while has no effects on the H3K4 trimethylation (H3K4me3) or H3K9 trimethylation (H3K9me3).

Key words： Plasmodium falciparum histone-lysine N-methyltransferase recombinant protein expression wheat germ cell-free expression system activity identification

收稿日期: 2016-07-01

基金资助:浙江省科技厅公益技术研究社会发展项目（2015C33101）；浙江省大学生科技创新活动计划（2014R 413079）；温州市公益科技项目（Y20140481）。

通讯作者: 梁韶晖，教授，硕士生导师，Email：lsh@wmu.edu.cn。

作者简介: 张亮亮（1989-），男，山西长治人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2017/V47/I2/110

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