FRACTIONATION PROTOCOL FOR THE ISOLATION OF POLYPEPTIDES FROM PLANT BIOMASS

A fractionation protocol for the isolation of a highly purified polype ptide fraction from plant biomass is described. The procedure dereplic ates ubiquitous substance classes known to interfere with bioassays of ten used in natural product-based drug discovery programs. The protoco l involves pre-extraction with dichloromethane, extraction with ethano l(50%), removal of tannins with polyamide, removal of low-molecular-we ight components with size-exclusion chromatography over Sephadex G-10, and final removal of salts and polysaccharides with solid-phase extra ction using reversed-phase cartridges. The method has been applied to the aerial parts of Viola arvensis, resulting in the isolation of a pe ptide fraction that on further separation yielded a novel 29-residue m acrocyclic polypeptide named vary peptide A, cyclo(-TCVGGTCNTPGCSCSWPV CTRNGLPVCGE-).