赤红球菌转录因子DevR的DNA结合活性分析  

Heterologous expression and DNA binding activity analysis of transcription factor DevR in Rhodococcus ruber

作　　者：李秋月 彭仁[1] LI Qiuyue;PENG Ren(College of Life Science,Jiangxi Normal University,Nanchang 330022,China)

机构地区：[1]江西师范大学生命科学学院,江西南昌330022

出　　处：《生物技术》2025年第1期6-13,共8页Biotechnology

基　　金：国家自然科学基金-地区科学基金项目(32160011)。

摘　　要：[目的]构建重组质粒pET-21b-devr,表达并纯化转录因子DevR,并进行DNA结合活性分析。[方法]对赤红球菌的转录因子DevR进行生物信息学分析,并对devr基因进行密码子优化,构建重组表达质粒pET-21b-devr,导入到大肠杆菌BL21(DE3)中进行异源表达,利用SDS-PAGE和Western Blotting检测表达情况。采用包涵体结合镍离子亲和层析的方法进行蛋白纯化,然后对其DNA结合活性进行分析。[结果]ProtParam工具分析表明DevR蛋白由216个氨基酸残基组成,分子量为23401.99 Da,亲水性总平均值为-0.071,含有一个REC结构域和一个LuxR型HTH结构域。在25℃、0.8 mmol/L的IPTG诱导条件下,重组蛋白DevR表达量最大,体外EMSA证实了转录因子DevR具有DNA结合活性。[结论]成功获得纯度>95%、浓度>1 mg/mL的DevR重组蛋白,并证明其具有DNA结合活性,为进一步揭示DevR与赤红球菌有机溶剂耐受性之间的关系奠定基础。[Objective]Recombinant plasmid pET-21b-devr was constructed.Expression and purification of transcription factor DevR was performed and its DNA-binding activity was analyzed.[Method]The transcription factor DevR of R.ruber was analyzed using bioinformatics.The devr gene was codon-optimized and the recombinant expression plasmid pET-21b-devr was constructed,which was introduced into Escherichia coli BL21(DE3)for heterologous expression.SDS-PAGE and Western Blotting were used to detect its expression.The protein was purified by inclusion body combined with nickel ion affinity chromatography,and its DNA binding activity was analyzed.[Result]Protparam ananlysis showed that DevR protein was composed of 216 amino acid residues.Its molecular weight was 23401.99 Da and the total average hydrophilicity was-0.071,containing a REC domain and a LuxR type HTH domainand.It was found that the maximum expression of the protein was achieved at 25℃and IPTG concentration of 0.8 mmol/L.The DNA-binding activity of transcription factor DevR was confirmed by EMSA in vitro.[Conclusion]DevR with a purity of>95%and concentration>1 mg/mL was successfully obtained and its DNA-binding activity were demonstrated,which laid the foundation for further revealing the relationship between DevR and organic solvent tolerance of Rhodococcus ruber.

关 键 词：赤红球菌 生物信息学 转录因子 重组质粒 异源表达 蛋白纯化 EMSA DNA结合活性 

分 类 号：Q936[生物学—微生物学]