Superdex s200

Purified MTA1:RBBP4, HDAC1:MTA1 and MTA1:HDAC1:RBBP4 complexes that had been gel filtrated were concentrated to 1 mg/ml and reapplied to the Superdex S200 column (GE Healthcare, UK). The mass of each protein complex was calculated immediately on elution with an Optilab T-rEX differential Refractive Index detector coupled to a DAWN HELEOS MALS detector (Wyatt Technology).

Amylose V (0.6% w v^-1) was incubated with purified GtfD (0.2 mg) under the conditions described in “Substrate specificity of P. beijingensis GtfD”. After incubation for 24 h at 37°C, the reaction was stopped by transfer to 100°C for 8 min. The HMM and LMM polysaccharide fractions were isolated by size-exclusion chromatography on a Superdex S-200 (10 x 300 mm; GE-Healthcare) using 25 mM ammonium bicarbonate as eluent at a flow rate of 0.5 ml min^-1. For comparison the amylose-derived A. chroococcum GtfD polymer was also produced and isolated as described before [15 ].

To ensure efficient incorporation of seleno-methionine into the expressed protein, B834 (DE3) cells (Novagen) were used. An overnight culture was grown at 37°C in LB. Unlabelled media (Molecular Dimesions) was inoculated with 10ml of overnight culture and grown until OD₆₀₀ reached 0.5. Cells were harvested and washed three times in PBS before resuspending the pellets in seleno-methionine labelled media (Molecular Dimension). After a 40 minute incubation at 20°C, cells were induced with 0.4mM IPTG and left to express for 16 hours.
Harvested cells were lysed using a microfluidizer at 20,000 psi in a buffer containing 50mM Tris pH 8.0, 300mM NaCl, and 5mM BME. Cell lysates were clarified by centrifugation at 20,000xg and filtered through a 0.45μm filter. Lysates were then loaded onto a HisTrap column (GE Healthcare) before washing with 20mM imidazole. The protein was eluted with a linear gradient of imidazole from 0-300mM. Fractions containing protein were pooled and dialysed overnight at 4°C in the presence of TEV in a buffer containing 20mM Tris pH 8.0, 150mM NaCl, 5mM imidazole, and 2mM BME. The following day, proteins were passed over a HisTrap column and the flowthrough collected which was concentrated and passed over a Superdex S200 (GE Healthcare) column equilibrated in 10mM Tris pH7.5, 50mM NaCl and 2mM DTT.

Analytical gel filtration experiments for the detection of protein–protein interactions were carried out using an analytical Superdex S200 (GE) gel filtration column with a flow rate of 0.5 ml/min on an Äkta purifier system (GEHealthcare) in a 50 mM Hepes pH 7.5, 300 mM KCl buffer at 20°C. The protein concentrations were 20 µM and the total applied sample volume was 100 µl in all cases. Protein elution was followed by recording the UV adsorption at 280 nm.