A novel recombinant fusion toxin protein immunosuppressive B7-2-PE40KDEL Purification.docVIP

 PAGE \* MERGEFORMAT 28 A novel recombinant fusion toxin protein immunosuppressive B7-2-PE40KDEL Purification Author: Guan Hai-Rong, Yu-Ying Sun, Zhi-Hong Yuan, Zhang Huili, Liang Fei, Liu Nan, Guo Si Kai, Xi Choi Ha, XI Yongzhi 【Abstract】 In order to establish an efficient shortcut to a large number of re-separation of B7-2-PE40KDEL line downstream purification of fusion proteins to compare different combinations of purification strategies and conditions, including reversed-phase chromatography, metal chelate chromatography, anion-exchange chromatography, blue dye affinity chromatography and gel filtration chromatography, using MTT assay targeted the fusion protein cytotoxic activity. The results showed that the hydrophobic phase reversed-phase chromatography were used in the conventional methanol and acetonitrile, the result is the separation of the recombinant fusion protein had occurred in the variability, separated from the column on the generated flocculent precipitate. In the blue dye affinity chromatography separation step, because the whole bacterial proteins with affinity resin, the composition took place more non-specific binding, is extremely difficult to distinguish between the target protein and miscellaneous proteins. However, PRSETA-B7-2-PE40KDEL expression vector with a translation-enhancing sequence of T7-g10, can express the target protein N terminal fusion 6 histidine, which is conducive to through the Ni-NTA Metal Chelate chromatography method for rapid High-purity in purified expression products. According to this feature, after repeated experimental design finalized the selection of metal chelate chromatography, anion-exchange chromatography and gel filtration chromatography purification of three-step route for the purification of separation of B7-2-PE40KDEL fusion protein. The purpose of the purity of protein obtained up to 95% and the total recovery rate of 8%. Western-blot experiments showed that protein can be obtained w