Analysis to identify the receptor for cholix toxin from Vibrio cholerae

2022-23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分析。进一步，为了分析CRP与重组霍乱毒素(RChxA)的体外结合作用，并在发现CRP与rChxA结合的基础上进行了X射线结晶学结构研究。将组氨酸标记的CRP基因克隆到pFASTBac1中，纯化重组杆状病毒，转化果蝇Sf9细胞产生重组杆状病毒颗粒。用杆状病毒感染Sf9细胞，表达组氨酸标记的CRP。用抗CRP或抗His标记抗体的Western blotting证实组氨酸标记的CRP的表达。在分析溶解度时，组氨酸标记的CRP是不溶的，只能在颗粒组分中检测到。因此，组氨酸标记的CRP不能用亲和层析进行纯化。由于标记了组氨酸的CRP不能被纯化，所以购买了商业上可用的CRP。没有完整的C反应蛋白，因此购买了C反应蛋白的胞外区。RChxA与C反应蛋白胞外区的结合和重叠分析。在毒素覆盖或结合试验中均未观察到结合。这可能表明，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.