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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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

Received: 01 July 2016

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	ZHANG Liangliang
	CAI Liya
	WEI Qimei
	JIANG Lubin
	LIANG Shaohui.

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https://xb.wmu.edu.cn/EN/ OR https://xb.wmu.edu.cn/EN/Y2017/V47/I2/110

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