Analysis to identify the receptor for cholix toxin from Vibrio cholerae

2022- 2023财年的目标是进行组氨酸标记的重组候选受体蛋白（cRP）的表达和纯化，用于毒素覆盖或结合试验。为了进一步分析cRP与重组胆毒素（rChxA）的体外结合，并利用X射线晶体学方法研究cRP与rChxA的结合情况，将组氨酸标记的cRP基因克隆到pFASTBac 1中，纯化重组杆状病毒，转化草地贪夜蛾（Spodoptera frugiperda，Sf 9）细胞，获得重组杆状病毒颗粒。用杆状病毒感染Sf 9细胞以表达组氨酸标记的cRP。使用抗cRP或抗His标签抗体通过蛋白质印迹法确认组氨酸标签cRP的表达。当分析溶解度时，组氨酸标记的cRP是不溶的，并且只能在沉淀级分中检测到。因此，组氨酸标记的cRP不能使用亲和色谱法纯化，因为组氨酸标记的cRP不能纯化，所以购买市售cRP。无法获得完整的cRP，因此购买了cRP的细胞外结构域。在rChxA和cRP的胞外结构域之间进行结合和覆盖测定。在毒素覆盖或结合试验中均未观察到结合。这可能提示cRP的胞外区不能与rChxA结合。

The objectives for the fiscal year 2022-23 were to conduct expression and purification of histidine-tagged recombinant candidate receptor protein (cRP) for use in toxin overlay or binding assays. Further, to analyze in vitro binding between cRP and recombinant cholix toxin (rChxA) and perform structural studies using X-ray crystallography upon finding binding between cRP and rChxA.Histidine-tagged cRP-gene was cloned in pFASTBac1, recombinant bacmids were purified and Spodoptera frugiperda (Sf9) cells were transformed to generate recombinant baculovirus particles. The Sf9 cells were infected with baculovirus to express the histidine-tagged cRP. The expression of histidine-tagged cRP was confirmed by Western blotting using anti-cRP or anti-His-tag antibodies. When analyzed for the solubility, the histidine-tagged cRP was insoluble and could only be detected in pellet fraction. Thus, histidine-tagged cRP could not be purified using affinity chromatography.As the histidine-tagged cRP could not be purified, commercially available cRP were purchased. Whole cRP was not available and therefore extra-cellular domain of cRP was purchased. Binding and overlay assays were carried out between rChxA and extra-cellular domain of cRP. No binding could be observed either in toxin overlay or binding assays. This might suggest that extra-cellular domain of cRP could not bind to rChxA.