Esf af culture media

Human RS1 was expressed and secreted from stably transformed Sf21 insect cells grown in culture for 96 hours [14 (link)]. Anion exchange and galactose affinity chromatography were used to purify RS1 from 1 litre of ESF-AF culture media (Expression Systems) as previously described [14 (link)]. An additional size exclusion chromatography (SEC) step was introduced after the galactose affinity chromatography as follows. Protein eluted from the galactose agarose column in 1M IPTG, 100mM sodium chloride and 20mM Tris-HCl, pH 7.5, was concentrated to 500μl, loaded on to a Superdex 200 10/300 column (GE Lifesciences), and run at 0.5ml/min flow rate controlled with an AKTA Pure FPLC. The column was buffered with 20mM Tris-HCl, pH 7.5, and 100mM NaCl. After pooling the peak fractions, the protein was concentrated to 0.1mg/ml using centrifugal filters with a mass cut-off of 10,000 kDa. Protein standards (GE lifesciences HMW kit) were run on the same column in the same buffer for molecular mass calibration.