犬尿氨酸-3-单加氧酶SibC的体外蛋白表达纯化与生化反应  

In vitro expression,purification and biochemical reaction of kynurenine 3-monooxygenase SibC

作　　者：姚紫薇 孙长利 李青连[1] 鞠建华[1,2] YAO Zi-wei;SUN Chang-li;LI Qing-lian;JU Jian-hua(CAS Key Laboratory of Marine Materia Medica,RNAM center for Marine Microbiology,Chinese Academy of Sciences,South China Sea Institute of Oceanology,Guangzhou 510301,China;College of Oceanology,University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院南海海洋研究所中国科学院热带海洋生物资源与生态重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物研究中心,广东广州510301 [2]中国科学院大学海洋学院,北京100049

出　　处：《中国海洋药物》2022年第1期17-23,共7页Chinese Journal of Marine Drugs

基　　金：国家自然科学基金项目(22037006);国家重点研发计划项目(2019YFC0312500);广东省促进经济高质量发展专项项目(2020032);广东省珠江人才计划创新团队项目(2019BT02Y262);南方海洋科学与工程广东省实验室引进人才团队重点专项项目(GML2019ZD0406)资助。

摘　　要：目的通过体外基因合成西比罗霉素(sibiromycin)生物合成途径中推测但未验证功能的犬尿氨酸-3-单加氧酶编码基因sibC(GenBank:FJ768674.1:1380-2810),研究该犬尿氨酸-3-单加氧酶SibC的体外生化功能,为解析放线菌素D中相关结构单元的生物合成途径提供参考。方法通过生物信息学分析体外全合成基因sibC的序列并克隆至表达载体,将重组质粒pET28a-sibC转化至E.coli BL21(DE3)中进行表达,利用Ni-NTA亲和层析法纯化SibC,以犬尿氨酸(kynurenine,kyn,1)为底物进行SibC的体外酶活性测试,利用高效液相色谱(HPLC)对酶反应产物进行分析。结果PCR验证证明表达菌株E.coli BL21(DE3)/pET28a/sibC构建成功,SibC蛋白在E.coli BL21(DE3)中获得可溶性表达,经Ni-NTA亲和层析法纯化获得SibC蛋白,纯化的SibC能够催化犬尿氨酸生成3-羟基犬尿氨酸(3-hydroxykynurenine,3-HK,2)。结论体外验证了sibiromycin中SibC的功能为犬尿氨酸-3-羟化酶,为进一步研究犬尿氨酸途径及放线菌素的生物合成奠定了基础。Objective To characterize the function of sibC(GenBank:FJ768674.1:1380-2810)in the biosynthetic gene cluster of sibiromycin in vitro,thus providing valuable clue to elucidate the biosynthetic pathway of the similar structural unit in actinomycin D pathway.Methods The sibC gene was chemically synthesized and transferred into plasmid pET28a to yield pET28a-sibC.The plasmid pET28a-sibC was transformed into E.coli BL21(DE3)for overexpression.SibC was purified by Ni-NTA affinity chromatography.Then kynurenine(kyn,1)was used as substrate for enzyme activity detection in vitro,and the enzyme reaction products were analyzed by high performance liquid chromatography(HPLC).Results PCR analysis verified that the expression strain E.coli BL21(DE3)/pET28a/sibC was successfully constructed,the SibC was produced in E.coli BL21(DE3)and purified to homogeneity in soluble form.The purified SibC catalysed the production of kynurenine to 3-hydroxykynurenine(3-HK,2)efficiently.Conclusion The function of SibC in sibiromycin biosynthetic pathway was validated in vitro,catalyzing the transformation from kynurenine to 3-hydroxykynurenine,laying the foundation for in depth study on the biosynthesis of marine derived actinomycin D.

关 键 词：放线菌素D 生物合成 犬尿氨酸途径 犬尿氨酸-3-单加氧酶 色氨酸代谢 

分 类 号：R961[医药卫生—药理学]