摘要
目的 RNF2是一种转录抑制因子,其N端保守的RING结构域使其能够发挥E3泛素连接酶的功能。本研究采用生物信息学工具对人源RNF2蛋白进行序列和结构分析,同时在大肠埃希菌表达系统中进行高效表达与纯化,为后续探索其生物学功能和潜在的药物靶点奠定基础。方法 利用ClustalX、Expasy、TMHMM及SignalP等软件对RNF2进行多序列比对、理化性质分析、跨膜区、信号肽及疏水性分析。通过分子克隆构建pGEX-4T-RNF2重组质粒,随后转入大肠埃希菌BL21(DE3)中进行目的蛋白的诱导表达。收集菌体后通过高压破碎释放重组蛋白,进一步使用GST亲和层析柱纯化。结果 RNF2全长共336个氨基酸,带负电,无跨膜区,无信号肽,属于亲水蛋白,且在不同物种中较为保守。成功构建了pGEX-4T-RNF2重组菌株,经IPTG诱导表达后,发现RNF2能够在BL21(DE3)中大量表达。GST层析柱纯化结果显示RNF2的挂柱效果较差,但仍能得到少量高纯度蛋白。结论 使用大肠埃希菌原核表达系统成功获得性状较好的RNF2重组蛋白,为后续功能实验和药物开发奠定了基础。
Objective RNF2 is a transcriptional repressor whose N-terminal conserved RING structural domain enables it to function as an E3 ubiquitin ligase.In this study,we used bioinformatics tools to analyze the sequence and structure of the human RNF2 protein.Additionally,we aim to achieve efficient expression and purification of the protein in an Escherichia coli expression system,laying the foundation for the subsequent exploration of its biological functions and potential drug targets.Methods Bioinformatics software such as ClustalX,Expasy,TMHMM,and SignalP were used to conduct multiple sequence alignment,physicochemical property analysis,transmembrane region analysis,signal peptide prediction,and hydrophobicity analysis of RNF2.The pGEX-4T-RNF2 recombinant plasmid was constructed by molecular cloning and subsequently transferred into E.coli BL21(DE3)for induced expression of the target protein.The recombinant protein was released through high-pressure disruption and further purified using a GST affinity chromatography column.Results RNF2 has a total length of 336 amino acids,is negatively charged,lacks transmembrane region and signal peptide.It is a hydrophilic protein,and is more conserved among different species.The pGEX-4T-RNF2 recombinant strain was successfully constructed.After IPTG-induced expression,it was found that RNF2 was able to be expressed in large quantities in BL21(DE3).The purification result of GST chromatography column showed that the hanging effect of RNF2 was poor,but a small amount of high-purity protein could still be obtained.The Western Blot results showed that RNF2 protein can be recognized by its specific antibody..ConclusionThe use of E.coli prokaryotic expression system successfully obtained the recombinant protein of RNF2 with better traits,which lays the foundation for the subsequent functional experiments and drug development.
作者
王琦
张俊梅
朱文菊
谢环环
解晓曼
赵桂华
徐超
尹昆
孙航
董宏杰
WANG Qi;ZHANG Junmei;XIE Huanhuan;ZHU Wenju;SUN Hang;XIE Xiaoman;ZHAO Guihua;YIN Kun;DONG Hongjie(Shandong Institute of Parasitic Diseases,Shandong First Medical University&Shandong Academy of Medical Sciences,Jining 272033,Shandong,China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2024年第10期1129-1135,共7页
Journal of Pathogen Biology
基金
山东省自然科学基金青年基金项目(No.ZR2023QH253)
山东省医药卫生科技发展计划(No.202201060464,202201050466,202201050463)
济宁市重点研发计划(2022YXNS158)
山东省高等学校“青创团队发展计划”(No.2022KJ194)
山东省泰山学者项目工程(No.tsqn202103186)
山东省医学科学院医药卫生科技创新工程。
关键词
RNF2
泛素化
异源表达
GST亲和层析
重组蛋白
RNF2
ubiquitination
heterologous expression
GST affinity chromatography
recombinant protein
作者简介
通讯作者:孙航,E-mail:sunhang1997@163.com;通讯作者:董宏杰,E-mail:donghongjie@sdfmu.edu.cn;王琦(1997-),女,山东人,硕士,研究实习员。主要研究方向:病原微生物重要致病因子的结构和功能研究。E-mail:15963743209@163.com。
