Soluble cell lysate was applied to the column and then extensively washed with Buffer A. MK2 was eluted from the column with Buffer A 40 mM glutath ione. The GST tag was cleaved using TEV protease, www.selleckchem.com/products/Romidepsin-FK228.html typi cally 4 16 h at 4 15 C. The sample was then diluted ten fold with Buffer B and loaded onto a MonoS 10 10 column equilibrated with Buffer B 20 mM NaCl. The protein was eluted at 200 mM NaCl using a stepwise Buffer B NaCl gradient. MK2 containing fractions were pooled and con centrated to 10 mg mL, and then loaded onto a Superdex 75 16 60 column equilibrated with 50 mM TrisHCl, 250 mM NaCl, 10% glycerol, 1 mM DTT, pH 7. 5. protein was eluted at 1 mL min. MK2 containing fractions were pooled and sample purity was assessed by SDS PAGE. protein identity was confirmed using mass spectrometry.
Activity Assays Enzymatic assays utilized a homogeneous time resolved fluorescence method to quantitate prod uct formation. Reactions contained in 40 L varying amounts of enzyme, 4 M peptide substrate, 10 M ATP, 20 mM MOPS, 10 mM MgCl2, 5 mM EGTA, 5 mM 2 phos phoglycerol, Inhibitors,Modulators,Libraries 1 mM Na3VO4, 0. 01% Triton X 100, 5% DMSO, 1 mM DTT, pH 7. 2. After 60 min at room temper ature, reactions were quenched by adding 10 L of 0. 5 M EDTA. Phosphorylated peptide was measured by addition of 75 L of 24 ng mL Eu3 cryptate labeled anti phospho 14 3 3 binding motif, 1. 47 g mL Sure Light allophycocyanin streptavidin, 50 mM HEPES, 0. 1% BSA, 0. 01% Tween 20, 0. 4 M KF. The developed reaction was incubated in the dark at room temperature for 10 min, then read in a time resolved flu orescence detector at 620 nm and 665 nm simultaneously, using a 337 nm nitrogen laser for excitation.
The 665 620 emis sion ratio is proportional Inhibitors,Modulators,Libraries to the amount of phosphor Inhibitors,Modulators,Libraries ylated peptide product. Since the HTRF method does not provide absolute quantities of product formed, specific activities were calculated as HTRF counts nM MK2 pro tein. Crystallization MK2 constructs yielding more than 1 mg L of culture were progressed Inhibitors,Modulators,Libraries to crystallization trials. Protein was concen trated to 10 mg mL using an Ultrafree 15 Biomax 10 kDa molecular weight cut off centrifugal Inhibitors,Modulators,Libraries filter device. Various inhibitors were added individually to con centrated protein stocks to a final concentration of 1 mM. Complexed MK2 protein was mixed with 0. 5 L of various crystallization solutions from Crystal Screen 1 and Crystal Screen 2 and Wizard Screen 1 and Wizard Screen 2.
The resulting drops were dispensed into 96 well sitting drop trays using a Hydra II 1 liquid handler. Trays were stored at 18 C and visualized manually. Crystallization was tested exten sively at 4 C, uniformly without success. Accumulated MK2 crystallization hits suggested parame ters to be explored more closely in a customized robotic screen, the most effective being the precipitating things reagent and pH range.