Methods for crystallizing ERK2 polypeptides
View Patent ↗The present invention relates to crystals of the ERK2 polypeptide and complexes thereof which are useful, inter alia, for structure assisted drug design.
1. A method for crystallizing a polypeptide comprising placing a non-liganded, non-phosphorylated polypeptide comprising SEQ ID NO:5 at a concentration of about 5 mg/mL to about 25 mg/mL in an aqueous buffered solution comprising 100 mM MES at a pH of from about 5.5 to about 6.7, from about 1% to about 8% polyethylene glycol (PEG) 400 (v/v), and about 2.4 M ammonium sulfate, and crystallizing the non-liganded, non-phosphorylated polypeptide comprising SEQ ID NO:5.
2. The method of claim 1 wherein the non-liganded, non-phosphorylated polypeptide comprising SEQ ID NO:5 is crystallized by a method selected from the group consisting of hanging-drop vapor diffusion and sitting drop vapor diffusion.
3. A method for crystallizing a polypeptide comprising placing a non-liganded, diphosphorylated or dithiophosphorylated polypeptide comprising SEQ ID NO:5, wherein Thr190 and Tyr192 of SEQ ID NO:5 are phosphorylated or wherein Thr190 and Tyr192 of SEQ ID NO:5 are thiophosphorylated, at a concentration of about 3 mg/mL to about 25 mg/mL in an aqueous buffered solution comprising 10 mM sodium citrate at a pH of from about 5 to about 6.2 and a concentration of from about 5% to about 30% isopropanol (v/v), and crystallizing the non-liganded, dephosphorylated or dithiophosphorylated polypeptide comprising SEQ ID NO:5.
4. The method of claim 3 wherein the non-liganded, diphosphorylated or dithiophosphorylated polypeptide comprising SEQ ID NO:5 is crystallized by a method selected from the group consisting of hanging-drop vapor diffusion and sitting drop vapor diffusion.
5. A method for soaking a ligand into a crystallized polypeptide comprising placing a non-liganded, non-phosphorylated polypeptide comprising SEQ ID NO:5 at a concentration of about 5 mg/mL to about 25 mg/mL in an aqueous buffered solution comprising 100 mM MES at a pH of from about 5.5 to about 6.7, from about 1% to about 8% polyethylene glycol (PEG) 400 (v/v), and about 2.4 M ammonium sulfate, and crystallizing the non-liganded, non-phosphorylated polypeptide comprising SEQ ID NO:5 by hanging drop vapor diffusion method and soaking said crystal in a solution comprising olomoucine, thereby soaking olomoucine into the crystallized non-liganded, non-phosphorylated polypeptide comprising SEQ ID NO:5.