EC Number   |
|---|
   3.1.13.4 | - |
| 3.1.13.4 | by metal affinity resin |
| 3.1.13.4 | by Ni2+ affinity chromatography and gel filtration, purity above 98% |
| 3.1.13.4 | Cell lysis is performed in a buffer containing 400 mM NaCl, 50 mM Tris/HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid and 2 mM dithiothreitol. The supernatant of the lysate is loaded onto a glutathione-Sepharose column and is eluted with lysis buffer containing 30 mM reduced glutathione. Glutathione S-transferase-PARN-RNA-recognition motif is incubated with protease and a final gel filtration is performed using a buffer containing 300 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 2 mM DTT. PARN-RNA-recognition motif is concentrated to 8.8 mg/ml using a vivaspin concentrator, and 7-methylguanosine triphosphate is added in 6fold molar excess. SeMet-containing PARN-RNA-recognition motif purification is analogous, with the exception that the DTT concentration is elevated to 5 mM. |
| 3.1.13.4 | His-tag affinity chromatography HiTrap Q HP and 7-Me-GTP-Sepharose affinity chromatography |
| 3.1.13.4 | Ni2+ matrix |
| 3.1.13.4 | of the recombinant protein |
| 3.1.13.4 | on Ni-NTA resin and by gel filtration, purity above 98% |
| 3.1.13.4 | overview on conditions |
| 3.1.13.4 | PARNn purified by glutathione-Sepharose 4B, MonoQ and Superdex 200 gel filtration columns. truncated PARN including the putative cap-binding domain purified by TALON affinity resin. full length PARN purified by Ni-NTA column |
