Maturated RumC1 obtained by heterologous coexpression of rumC1 and rumMc1 genes in E. coli and purified as described above was treated with either RumPc or TPCK (N-tosyl-l-phenylalanine chloromethyl ketone)–treated trypsin (Sigma-Aldrich) for 1 hour at 37°C. The molar ratios used were 200:1 for mRumC1:trypsin and 1:5 for mRumC1:RumPc. mRumC1c and mRumC1cc were purified using RP-C18-HPLC with the following gradient: 10 min at 22% followed by 12 min from 22 to 38% of 90% ACN and 0.1% TFA. For the preparation of large amounts of mRumC1cc for biological activity assays, mRumC1 was cleaved with trypsin and purified with the above RP-C18-HPLC conditions on a preparative column (250 mm by 21.2 mm; Phenomenex, Jupiter, 15 μm, 300 Å).

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