|本期目录/Table of Contents|

[1]刘瑞龙,向嵩.FBH1的原核表达及纯化方法的建立[J].天津医科大学学报,2026,32(04):357-361.[doi:10.20135/j.issn.1006-8147.2026.04.0357]
　LIU Ruilong,XIANG Song.Establishment of prokaryotic expression and purification method for FBH1[J].Journal of Tianjin Medical University,2026,32(04):357-361.[doi:10.20135/j.issn.1006-8147.2026.04.0357]
点击复制

FBH1的原核表达及纯化方法的建立(PDF)

《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
32卷
期数:
2026年04期
页码:
357-361
栏目:
论著
出版日期:
2026-07-10

文章信息/Info

Title:
Establishment of prokaryotic expression and purification method for FBH1
文章编号:
1006-8147（2026）04-0357-05
作者:
刘瑞龙; 向嵩
（天津医科大学基础医学院生物化学与分子生物学系，天津 300070）
Author(s):
LIU Ruilong;  XIANG Song
（Department of Biochemistry and Molecular Biology， School of Basic Medical Sciences， Tianjin Medical University， Tianjin 300070， China）
关键词:
FBH1; 蛋白质纯化; ATP水解; DNA结合
Keywords:
FBH1;  protein purification;  ATP hydrolysis;  DNA binding
分类号:
Q5
DOI:
10.20135/j.issn.1006-8147.2026.04.0357
文献标志码:
A
摘要:
目的：建立一种高效的FBH1的原核表达和纯化方法，并在体外验证其活性。方法：将密码子偏好性修改的人源FBH1基因插入到pMAL-c2x载体中。将重组质粒转化至大肠埃希菌Rosetta菌株中。随后采用异丙基-β-D-硫代半乳糖苷诱导FBH1蛋白表达。采用亲和层析、离子交换层析和凝胶过滤层析纯化FBH1。通过ATP水解实验和凝胶迁移实验验证纯化的FBH1蛋白活性。结果：建立了FBH1全长在大肠杆菌中大量表达的方法，并进一步通过亲和、离子交换和分子筛层析纯化的方法精细纯化，获得了无核酸污染，纯度为40%以上的FBH1蛋白，并且通过体外功能实验证明了制备的人源重组蛋白FBH1具有被报道的生化活性，包括能够与复制叉结合和ATP水解的活性。结论：成功建立了从大肠埃希菌中制备人源FBH1的方案，获得较大量的FBH1蛋白，并且通过功能实验证明了所制备的FBH1具有期望的生化活性。
Abstract:
Objective： To establish an efficient prokaryotic expression and purification method for FBH1， and to validate its activity in vitro. Methods： A codon-optimized human FBH1 gene was inserted into the pMAL-c2x vector. The recombinant plasmid was transformed into the Rosetta strain of Escherichia coli. FBH1 protein expression was then induced with isopropyl-β-D-thiogalactoside （IPTG）. FBH1 was purified with affinity chromatography， ion exchange chromatography， and gel filtration chromatography. The activity of the purified FBH1 protein was verified through ATP hydrolysis assays and gel shift assays. Results： A method was established for the large-scale expression of full-length FBH1 in E. coli. The protein was further purified by affinity， ion exchange， and molecular sieve chromatography， yielding FBH1 protein that was free from nucleic acid contamination and had a purity over 40%. In vitro functional assays confirmed that the prepared human recombinant FBH1 exhibited the reported biochemical activities， including the ability to bind replication forks and catalyze ATP hydrolysis. Conclusion： This study successfully established a method to prepare human FBH1 from E. coli， obtaining a substantial amount of FBH1 protein. Functional assays demonstrates that the prepared FBH1 possesses the expected biochemical activity.

参考文献/References:

[1] BRANZEI D， FOIANI M. Maintaining genome stability at the replication fork[J]. Nat Rev Mol Cell Biol， 2010， 11（3）： 208-219.
[2] BERTI M， CORTEZ D， LOPES M. The plasticity of DNA replication forks in response to clinically relevant genotoxic stress[J]. Nat Rev Mol Cell Biol， 2020， 21（10）： 633-651.
[3] BERTI M， VINDIGNI A. Replication stress： getting back on track[J]. Nat Struct Mol Biol， 2016， 23（2）： 103-109.
[4] MACHERET M， HALAZONETIS T D. DNA replication stress as a hallmark of cancer[J]. Annu Rev Pathol， 2015， 10： 425-448.
[5] RICKMAN K， SMOGORZEWSKA A. Advances in understanding DNA processing and protection at stalled replication forks[J]. J Cell Biol， 2019， 218（4）： 1096-1107.
[6] FUGGER K， MISTRIK M， NEELSEN K J， et al. FBH1 Catalyzes regression of stalled replication forks[J]. Cell Rep， 2015， 10（10）： 1749-1757.
[7] FUGGER K， MISTRIK M， DANIELSEN J R， et al. Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities[J]. J Cell Biol， 2009， 186（5）： 655-663.
[8] POOLE L A， CORTEZ D. Functions of SMARCAL1， ZRANB3， and HLTF in maintaining genome stability[J]. Crit Rev Biochem Mol Biol， 2017， 52（6）： 696-714.
[9] HALDER S， RANJHA L， TAGLIALATELA A， et al. Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex[J]. Nucleic Acids Res， 2022， 50（14）： 8008-8022.
[10] BANSBACH C E， B?魪TOUS R， LOVEJOY C A， et al. The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks[J]. Genes Dev， 2009， 23（20）： 2405-2414.
[11] YUSUFZAI T， KADONAGA J T. HARP is an ATP-driven annealing helicase[J]. Science， 2008， 322（5902）： 748-750.
[12] YUSUFZAI T， KONG X， YOKOMORI K， et al. The annealing helicase HARP is recruited to DNA repair sites via an interaction with RPA[J]. Genes Dev， 2009， 23（20）： 2400-2404.
[13] LORENZ A， OSMAN F， FOLKYTE V， et al. Fbh1 limits Rad51-dependent recombination at blocked replication forks[J]. Mol Cell Biol， 2009， 29（17）： 4742-4756.
[14] ZEMAN M K， CIMPRICH K A. Causes and consequences of replication stress[J]. Nat Cell Biol， 2014， 16（1）： 2-9.
[15] FLAUS A， OWEN-HUGHES T. Mechanisms for ATP-dependent chromatin remodelling： the means to the end[J]. FEBS J， 2011， 278（19）： 3579-3595.
[16] HUANG C， GU S， YU P， et al. Deficiency of smarcal1 causes cell cycle arrest and developmental abnormalities in zebrafish[J]. Dev Biol， 2010， 339（1）： 89-100.
[17] CICCIA A， BREDEMEYER A L， SOWA M E， et al. The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart[J]. Genes Dev， 2009， 23（20）： 2415-2425.
[18] CICCIA A， NIMONKAR A V， HU Y， et al. Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress[J]. Mol Cell， 2012， 47（3）： 396-409.
[19] KIM H J， KIM H J. Yeast as an expression system for producing virus-like particles： what factors do we need to consider?[J]. Lett Appl Microbiol， 2017， 64（2）： 111-123.
[20] FRANCHITTO A， PICHIERRI P. Replication fork recovery and regulation of common fragile sites stability[J]. Cell Mol Life Sci， 2014， 71（23）： 4507-4517.
[21] KUZMINOV A. Chromosomal replication complexity： a novel DNA metrics and genome instability factor[J]. PLoS Genet， 2016， 12（10）： e1006229.

相似文献/References:

[1]龚晓昕,沈苗苗,李徐梓超,等.人源解旋酶样转录因子的表达、纯化和功能研究[J].天津医科大学学报,2022,28(01):35.
　GONG Xiao-xin,SHEN Miao-miao,LI Xu-zi-chao,et al.Expression，purification of human helicase-like transcription factor[J].Journal of Tianjin Medical University,2022,28(04):35.
[2]赵佳奇,石萌,向嵩.Bre1与Rad6形成的复合物的表达、纯化和结晶[J].天津医科大学学报,2023,29(01):83.
　ZHAO Jia-qi,SHI Meng,XIANG Song.Expression，purification and crystallization of Bre1-Rad6 complex[J].Journal of Tianjin Medical University,2023,29(04):83.

备注/Memo

备注/Memo:
（2025-12-29收稿）
基金项目 国家自然科学基金（32271259）
作者简介 刘瑞龙（2000-），男，硕士在读， 研究方向：生物学；通信作者：向嵩，E-mail：xiangsong@tmu.edu.cn。
更新日期/Last Update: 2026-07-15