Expression, purification, and characterization of the histidine kinase CarS from <i>Fusobacterium nucleatum</i>.

Zhuting LI; Xian Shi; Ruochen Fan; Lulu Wang; Tingting BU; Wei Zheng; Xuqiang Zhang; Chunshan Quan

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

Author: Zhuting LI ¹ ; Xian SHI ¹ ; Ruochen FAN ² ; Lulu WANG ² ; Tingting BU ¹ ; Wei ZHENG ¹ ; Xuqiang ZHANG ¹ ; Chunshan QUAN ¹
Author Information

1. Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, Dalian Minzu University, Dalian 116600, Liaoning, China.
2. School of Bioengineering, Dalian University of Technology, Dalian 116081, Liaoning, China.
Publication Type:Journal Article
Keywords: CarRS two-component system; Fusobacterium nucleatum; kinase activity; phosphotransferase activity
MeSH: Humans; Histidine Kinase/metabolism*; Fusobacterium nucleatum/metabolism*; Automobiles; Protein Kinases/genetics*; Escherichia coli/metabolism*; Colorectal Neoplasms
From: Chinese Journal of Biotechnology 2023;39(4):1596-1608
CountryChina
Language:Chinese
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.