人组蛋白α-氨基乙酰基转移酶Nat11表达纯化、晶体生长及底物结合研究

生物技术通报 ›› 2014, Vol. 0 ›› Issue (11): 193-200.

人组蛋白α-氨基乙酰基转移酶Nat11表达纯化、晶体生长及底物结合研究

黄嘉欣,李海涛

清华大学医学院基础医学系,北京 100086

收稿日期:2014-04-08 出版日期:2014-11-07 发布日期:2014-11-07
作者简介:黄嘉欣,女,硕士研究生,研究方向：结构表观遗传学
基金资助:
科技部“973”项目（2011CB965303）,2012教育部新世纪优秀人才支持计划

Expression, Crystallization and Substrate Binding Studies of Human Histone N-terminal Acetyltransferase Nat11

Huang Jiaxin ,Li Haitao

Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100086

Received:2014-04-08 Published:2014-11-07 Online:2014-11-07

摘要/Abstract

摘要： α-氨基乙酰基转移酶11（Nat11）催化组蛋白H4和H2A氨基端乙酰化修饰,发挥着重要的表观遗传调控功能。将人Nat11基因构建到原核表达载体pSUMO中,转化入大肠杆菌BL21（DE3）进行重组表达。通过镍柱亲和层析等一系列体外纯化步骤,获得高纯度Nat11。利用等温滴定量热法（ITC）,测得Nat11与底物多肽微摩尔量级结合常数。利用质谱技术,发现纯化后的Nat11结合有大肠杆菌内源产生的乙酰辅酶A或辅酶A,在ITC滴定过程中可以产生对多肽底物的乙酰化修饰,表明纯化获得的Nat11在溶液中具有酶活力。随后,对Nat11进行晶体生长研究,通过初筛优化获得蛋白截短体及底物-酶融合蛋白单晶。

关键词: α-氨基乙酰基转移酶11, 重组蛋白表达, 蛋白聚集, 酶-底物结合, 晶体生长

Abstract: The alpha-amino groups of histones H4 and H2A can be acetylated by histone N-terminal acetyltransferase 11（Nat11）, which plays an important role in epigenetic regulation. The cDNA of human Nat11 was amplified and cloned into pSUMO vector. The resultant construct was transformed into E.coli strain BL21（DE3）for recombinant protein expression. Homogenous Nat11 was highly purified through a series of purification procedures including nickel column affinity chromatography. Using isothermal titration calorimetry（ITC）, we measured micromolar binding constants between Nat11 and histone H4 peptides. MALDI-TOF mass spectrometry analysis revealed that purified Nat11 was pre-bound with acetyl coenzyme A or coenzyme A that was co-purified from E.coli. After ITC titration using unmodified peptide as ligand, N-acetylated product was detected by mass spectrometry, suggesting that the purified Nat11 is active. We performed crystallization screening and successfully obtained single crystal of a truncate form of Nat11 and substrate-enzyme recombinant protein after optimization.

Key words: Alpha-amino acetyltransferase 11 , Recombinant protein expression , Protein aggregation , Enzyme-substrate binding , Crystallization

黄嘉欣,李海涛. 人组蛋白α-氨基乙酰基转移酶Nat11表达纯化、晶体生长及底物结合研究[J]. 生物技术通报, 2014, 0(11): 193-200.

Huang Jiaxin ,Li Haitao. Expression, Crystallization and Substrate Binding Studies of Human Histone N-terminal Acetyltransferase Nat11[J]. Biotechnology Bulletin, 2014, 0(11): 193-200.

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