精子发生调控因子重组人HSF5的原核表达、活性检测与结构预测

基础医学与临床 ›› 2021, Vol. 41 ›› Issue (9): 1323-1328.

精子发生调控因子重组人HSF5的原核表达、活性检测与结构预测

钟洪莉¹, 肖城良², 史祥睿³, 代宇婕⁴, 刘威^4*, 张庆华^1*

1.陆军军医大学大坪医院妇产科,重庆 400042;
2.华中科技大学生命科学与技术学院,湖北武汉 430065;
3.沈阳化工大学化学工程学院, 辽宁沈阳 110021;
4.陆军军医大学全军免疫研究所, 重庆 400038

收稿日期:2021-02-09 修回日期:2021-06-15 出版日期:2021-09-05 发布日期:2021-09-02
通讯作者: *wei.liu.2005@gmail.com; zhangqh1123@163.com
基金资助:
重庆市社会民生科技创新专项(30118-2812)

Expression, activity measurement and structure prediction of spermatogenic regulator:recombinant human HSF5

ZHONG Hong-li¹, XIAO Cheng-liang², SHI Xiang-rui³, DAI Yu-jie⁴, LIU Wei^4*, ZHANG Qing-hua^1*

1. Department of Obstetrics and Gynecology, Daping Hospital, Army Medical University, Chongqing 400042;
2. College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430065;
3. College of Chemical Engineering, Shenyang University of Chemical Technology, Shenyang 110021;
4. Institute of Immunology, Army Medical University, Chongqing 400038,China

Received:2021-02-09 Revised:2021-06-15 Online:2021-09-05 Published:2021-09-02
Contact: *wei.liu.2005@gmail.com; zhangqh1123@163.com

摘要/Abstract

摘要： 目的表达可溶性重组人源热休克转录因子5(rhHSF5)蛋白,并对其与热休克元件(HSEs)结合的结构模型进行预测。方法将目的基因克隆到表达载体pET-22b(+)上,然后将重组质粒转化至大肠杆菌BL21(DE3)菌株中,挑取单克隆,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。通过Ni-NTA亲和层析、Superdex 75分子筛层析纯化后,获得重组蛋白。与包含HSEs的DNA片段进行凝胶迁移实验,并通过同源建模和分子动力学模拟研究方法对其与HSEs结合的结构进行模拟和预测。结果获得可溶性人源HSF5蛋白的表达;经纯化得到高纯度、可结合HSEs的重组蛋白;凝胶迁移实验可见它与含HSEs的寡聚核苷酸有结合。结论可溶性的人源HSF5蛋白可在原核系统中表达,为进一步研究HSF5的功能机制提供了基本条件。

关键词: 精子发生, 热休克转录因子5(HSF5), 原核表达, 可溶性蛋白, 结构预测

Abstract: Objective To obtain soluble expression of recombinant human heat shock transcription factor 5(rhHSF5) proteins and predict the structure of HSF5 bound to heat shock element. Methods The nucleotide sequence encoding the target gene was subcloned into expression vector pET-22b(+), followed by transformation into Escherichia coli BL21(DE3) strain and expression induction with IPTG. The rhHSF5 was purified using a Ni-NTA affinity column and gel filtration chromatography. A gel shift assay was carried out after the incubation of the purified rhHSF5 and an oligonucleotide containing a heat shock element. Structure prediction was done by means of homology modeling and molecular dynamics simulation. Results Soluble expression of rhHSF5 was achieved with high purity production. The purified rhHSF5 showed good binding activity with the heat shock element. A reasonable protein-DNA binding model was obtained. Conclusions The recombinant human HSF5 is produced in a soluble form with good yield and activity, which lays a solid foundation for mechanistic studies of this novel spermatogenesis regulator in the future.

Key words: spermatogenesis, heat shock transcription factor 5(HSF5), prokaryotic expression, soluble protein, structure prediction

中图分类号:

R318

钟洪莉, 肖城良, 史祥睿, 代宇婕, 刘威, 张庆华. 精子发生调控因子重组人HSF5的原核表达、活性检测与结构预测[J]. 基础医学与临床, 2021, 41(9): 1323-1328.

ZHONG Hong-li, XIAO Cheng-liang, SHI Xiang-rui, DAI Yu-jie, LIU Wei, ZHANG Qing-hua. Expression, activity measurement and structure prediction of spermatogenic regulator:recombinant human HSF5[J]. Basic & Clinical Medicine, 2021, 41(9): 1323-1328.

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