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find Author "LIYuan-bo" 1 results
  • Optimization of HSP65-MUC1 Purification in Pilot Scale and Identification of Methods to Detect Biological Function

    ObjectiveTo optimize HSP65-MUC1 fusion protein purification in pilot scale through protein purification techniques and identify the methods for biological activity detection. MethodsE. coli expressing HSP65-MUC1 was obtained by fermentation, then homogenized to obtain the supernatant. To acquire high-purity, high-quality HSP65-MUC1, the supernatant was treated with saturated ammonium sulfate, phenyl sepharose FF column and Q FF ion-exchange chromatography column purification. The expression of CD86 on the surface of DC cells treated with HSP65-MUC1 was determined with flow cytometry. ResultsE. coli containing pET28a-HSP65-MUC1 recombinant plasmid can effectively express target protein. A total of 413.7 mg of HSP65-MUC1 was obtained after 10 g of fermented cells was treated with saturated ammonium sulfate, phenyl sepharose FF column and Q FF ion-exchange chromatography column, and the purity was nearly 96%. Compared with negative control (10.13%±0.89%), purified HSP65-MUC1 could significantly improve the expression of CD86 on the surface of DC cells (29.98%±1.02%). ConclusionThe pilot scale production of purified HSP65-MUC1 has been effectively optimized, and the methods of its biological activity detection have been identified, which simultaneously provides the basis for clinical studies.

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