中国生物工程杂志

Objective:To establish the purification process of anti-IgE single chain antibody and identify its biological activity. Methods:Protein L was screened as the first step, and the nickel affinity chromatography was used as the second step of the purification according to the characteristics of protein L affinity chromatography and Ni affinity chromatography. The purification process of anti-IgE single chain antibody was established basing on the study of the loading and the eluting conditions of the two kinds of purification methods. Results:The purification process of protein L:pH=3.5 was the best pH value of washing impurity and pH=2.0 was the best pH value of eluting the target protein. The target protein was collected in the loading buffer of Ni affinity chromatography. The polishing process of Ni affinity chromatography was:Impurity was washed by 50mmol/L imidazole solution and the target protein was eluted by 500mmol/L imidazole solution. The purity of the target protein was 99.0% by SDS-PAGE electrophoresis analysis and 99.5% by CE-SDS electrophoresis analysis and the total yield came up to 80.0% after the two purification steps. The purified protein was detected by competitive ELISA and Biacore analysis, which confirmed that the product could recognize IgE specifically and its affinity was 8.59e^-9M,The product showed good neutralizing IgE activity by competitive Biacore analysis,its EC₅₀ which inhibited IgE was 70nM. Conclusions:An efficient and simple purification process of anti-IgE single chain antibody which was expressed in E.coli has been established and would be the basis of further scale-up process study. At the same time,it also proved that the novel small molecule anti-IgE antibody had good specificity and affinity to the target and displayed medical value. The purification technology of small molecule antibody could be insight to the purification of other small molecules.