人赖氨酸乙酰基转移酶7结构域蛋白的原核表达及纯化  

Prokaryotic Expression and Purification of Human KAT7 Domains

作　　者：黄俊[1] 麦海星[1] 徐小洁[2] 梁迎春[2] 尤文叶 李玲[2] 高风[3] 叶棋浓[2] 陈立军[1] 

机构地区：[1]军事医学科学院附属医院,北京100071 [2]军事医学科学院生物工程研究所,北京100850 [3]北京军区北戴河疗养院疗一科,河北秦皇岛066100

出　　处：《生物技术通讯》2016年第6期812-815,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81472589;81502264;81572597);北京市科技新星计划(Z141102001814055);北京市自然科学基金(7132155)

摘　　要：目的:克隆人赖氨酸乙酰基转移酶7(KAT7)的2个功能结构域基因,获得其原核表达产物,并纯化蛋白。方法:采用PCR技术从人乳腺文库中扩增人KAT7基因的2个功能结构域(1-330aa)和(331-611aa)编码片段,将其克隆到p ET28a载体中,在大肠杆菌Rossate中表达后,对原核表达产物进行纯化,以SDS-PAGE和Western印迹鉴定表达与纯化产物。结果:从人乳腺文库中分别扩增获得约990和840 bp的DNA片段,并克隆至p ET28a载体,测序结果表明与目的序列完全一致;在大肠杆菌Rossate中诱导表达出相对分子质量分别约为42 000和36 000的目的蛋白,经纯化后获得了纯度较高的重组蛋白KAT7(1-330aa)和(331-611aa)。结论:获得了重组蛋白KAT7(1-330aa)和(331-611aa),为后续研究KAT7与肿瘤调控奠定了实验基础。Objective： To construct the prokaryotic expression vector of human lysine acetyltransferases 7（KAT7）domains 1-330 aa and 331-611 aa, and obtain the purified protein. Methods： Human KAT7（1-330aa） and KAT7（331-611aa） coding region were amplified from human mammary c DNA library, and were inserted into prokaryoticexpression vector p ET28 a. The recombinant plasmid p ET28a- KAT7（1- 330aa） and p ET28a- KAT7（331- 611aa）were transformed into E.coli Rossate. The expressed products were purified and identified by SDS-PAGE and West-ern blot analysis. Results： The DNA fragments of about 990 and 840 bp were amplified by PCR, cloned intop ET28 a, and identified by sequencing. The recombinant proteins of about Mr 42 000 and 36 000 were induced,purified and verified by kinase assay. Conclusion： The domains of KAT7（1-330aa） and KAT7（331-611aa） wereobtained, which lay the foundation for further research on tumor.

关 键 词：人赖氨酸乙酰基转移酶7 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学]