PRMT3
PDB:4HSG
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:GI:44771198
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site:MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).
Construct
Prelude:
Sequence:DLQEDEDGVYFSSYGHYGIHEEMLKDKIRTESYRDFIYQNPHIFKDKVVLDVGCGTGILSMFAAKAGAKKVLGVDQSEILYQAMDIIRLNKLEDTITLIKGKIEEVHLPVEKVDVIISEWMGYFLLFESMLDSVLYAKNKYLAKGGSVYPDICTISLVAVSDVNKHADRIAFWDDVYGFKMSCMKKAVIPEAVVEVLDPKTLISEPCGIKHIDCHTTSISDLEFSSDFTLKITRTSMCTAIAGYFDIYFEKNCHNRVVFSTGPQSTKTHWKQTVFLLEKPFSVKAGEALKGKVTVHKNKKDPRSLTVTLTLNNSTQTYGLQ
Vector:pET28a-LIC
Growth
Medium:
Antibiotics:
Procedure:The overexpression of the recombinant protein in E. coli BL21 (DE3)pRARE2-V2R was induced by addition of 1 mM isopropyl-1-thio-D-galactopyranoside (IPTG) and overnight incubation at 15 oC.
Purification
Procedure
After clarification of the crude extract by centrifugation (16,000 rpm for onehour using Aventi-J 20-XPI, Beckman Coulter), the lysate was loaded ontoDE52 ion-exchange resin and passed through a Ni-NTA column. The columnwas washed and protein was eluted by addition of 20 mM Tris buffer, pH7.5, 500 mM NaCl, 5% glycerol, containing 30 mM and 250 mM imidazole,respectively. Protein sample was purified further using 6mL Resource QAnion exchange column (Code No:17-1179-01 , GE Healthcare) and ÄKTAFPLC (GE Healthcare) where the column was pre-equilibrated with 10 mMTris buffer, pH 7.6, followed by 10 mM Tris buffer, pH 7.6 with 1 M NaCland then re-equilibrated with 10 mM Tris buffer, pH 7.6 . The diluted protein sample (final NaCl concentration of 50 mM) was loaded onto a 6 mL ResourceQ column .The Contaminant proteins bind and pure PRMT3 would be in flow throw with an approximate purity of 95% purity. After adjusting the NaCl final concentration to 150 mM, the protein was concentrated, flash-frozen and storedat -80 oC.
Extraction
Procedure
Harvested cells were re-suspended in 20 mM Tris buffer, pH 7.5, with 500mM NaCl, 5 mM imidazole and 5% glycerol. The cells were lysed chemically followed by sonication at a frequency of 8.5 with ten seconds on and ten seconds off (Sonicator 3000, Misoni).
Concentration:Purified protein was concentrated using 15 mL concentrators with an appropriatemolecular weight cut-off (Amicon Ultra-15 10,000 MWCO, Millipore) to a final value of2.2 mg/mL.
Ligand
MassSpec:
Crystallization:PRMT3 was incubated at 1.1 mg/mL overnight 1-(1,2,3-benzothiadiazol-6-yl)-3-(2-oxo-2- phenylethyl)urea (compound 1) at 1:30 molar ratio (PRMT3: compound 1). Following incubation, protein was concentrated to 3 mg/mL andcrystallized using the sitting drop diffusion method at 20°C by mixing 1 µL ofthe protein solution with 1 µL of the reservoir solution containing 25% PEG 3350, 0.2 M Lithium Sulfate, 0.1 M HEPES, pH 7.5, vapor diffusion hanging drop, temperature 18°C . Prior to freezing, 0.1 µL of 100 mM compound 1 was added directly to the drop. Crystals were soaked for 30 min in the same buffer with 10% glycerol.
NMR Spectroscopy:
Data Collection:
Data Processing:
