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Microfiltration, ultrafiltration, cation exchange and size-exclusion chromatography were used to isolate FIP-fve from Flammulina velutipes fruiting body in the pilot scale. At the sample preparing stage, the homogenate of mushroom is usually hard to be resolved hence microfiltration was used to clarify the homogenate of F. velutipes efficiently. Next ultrafiltration is applied to concentrating supernatant and removal of small molecules. The samples from ultrafiltration can be directly used for the chromatography stage, and in simplifying the entire sample preparation process, all technical parameters can be amplified linearly. Concentrated supernatant from ultrafiltration was further separated with two-step chromatographic purification of strong cation-exchange and size-exclusion, purified product shows as single band at 13 kDa in SDS-PAGE gel, the analysis result of reversed phase HPLC C4 column also indicated that purified product has a great purity (95% or more). The purified sample was identified as FIP-fve (sequence coverage, more than 95%) by MALDI TOF/TOF, and its molecular weight was 12 740Da. The purification process is reproducible, robust and well suited for scaling-up to an industrial scale operation.

New purification process of fungal immunomodulatory protein, FIP-fve from Flammulina velutipes via filtration and column chromatography