具核梭杆菌CarRS双组分系统组氨酸激酶CarS的表达、纯化及生化活性测定

Expression, purification, and characterization of the histidine kinase CarS from Fusobacterium nucleatum

摘要:

具核梭杆菌(Fusobacterium nucleatum)是一种条件致病菌，能够在结直肠癌组织中富集，影响结直肠癌发生发展的多个阶段。双组分系统在病原菌耐药性、致病性相关基因的调控和表达中起重要作用。本文以具核梭杆菌CarRS双组分系统为研究对象，重点对其组氨酸激酶蛋白CarS进行重组表达和性质研究。利用在线软件SMART、CCTOP和AlphaFold2对CarS二级结构和三级结构进行预测，其结果表明CarS蛋白具有2个跨膜螺旋区，包含9个α螺旋和12个β折叠结构；由两个结构域构成，一是位于N末端的跨膜域(氨基酸1-170)，另一个是位于C末端的胞内域，胞内域由信号接收域(histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP)、磷酸受体结构域(histidine kinase domain, HisKA)和组氨酸激酶催化结构域(histidine kinase-like ATPase catalytic domain, HATPase_c)组成。由于全长的CarS蛋白未能在宿主细胞中表达，因此根据其二级、三级结构特点，构建了CarS胞内蛋白的融合表达载体pET-28a(+)-MBP- TEV-CarS_cyto，并在大肠杆菌(Escherichia coli) BL21-CodonPlus(DE3)-RIL中进行过表达。经亲和层析、离子交换层析和凝胶过滤层析，最终获得纯度较高的CarS_cyto-MBP蛋白，终浓度达20 mg/mL。活性实验结果表明，CarS_cyto-MBP具有蛋白激酶和磷酸转移酶双活性，麦芽糖结合蛋白(maltose binding protein, MBP)标签对CarS_cyto蛋白的生物活性无影响。上述结果为深入解析CarRS双组分系统在具核梭杆菌中的生物学功能提供了一定的理论基础。

关键词:具核梭杆菌;CarRS双组分系统;蛋白激酶活性;磷酸转移酶活性

Abstract:

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarS_cyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarS_cyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarS_cyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarS_cyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.

Key words:Fusobacterium nucleatum;CarRS two-component system;kinase activity;phosphotransferase activity