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2.4 Enzymatic cleavage to remove GST affinity tag Affinity-purified fusion protein Vector-specific enzyme (thrombin, factor Xa, or PreScission protease) 0.15 M PMSF in isopropanol PBS/EDTA/PMSF: 1X PBS, 5 mM EDTA, 0.15 mM PMSF, pH 7.4 2.5 Removal of GST affinity tag after protease cleavage Cleaved fusion protein dialysed into PBS/EDTA/PMSF Glutathione Sepharose column PBS/EDTA/PMSF 2.6 Further purification of cleaved recombinant protein Cleaved fusion protein PBS Concentrator (Amicon Ultra) HPLC system with in-line detector and fraction collector Gel filtration column compatible with the molecular weight range of the sample to be purified 3

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