Method for crystallizing aurora 2 kinase catalytic domain polypeptide
View Patent ↗The present invention discloses nucleic acids that encode an active human Aurora 2 kinase catalytic domain. The present invention also discloses methods of growing X-ray diffractable crystals of polypeptides comprising the active human Aurora 2 kinase catalytic domain. The present invention further discloses a crystalline form of a catalytic domain of human Aurora 2 kinase. In addition, the present invention discloses methods of using the X-ray diffractable crystals of human Aurora 2 kinase in structure assisted drug design to identify compounds that can modulate the enzymatic activity of human Aurora 2 kinase.
1. A method for obtaining a crystal suitable for ligand exchange, comprising incubating a polypeptide at a concentration of about 15-25 mg/ml in a buffered solution, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 13 and wherein the buffered solution is at a pH range of 6.0-6.9 and comprises 5-24% polyethylene glycol 5000 monomethyl ether (MPEG 5000), 0-20% polyethylene glycol 2000 dimethyl ether (PEG 2000), 0.1 _M 2-(N-morpholino)ethanesulfonic acid (MES), 0.002 _M Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), and 0.05 to 0.4 M ammonium sulfate.
2. The method of claim 1 , wherein the buffered solution is at a pH of 6.5 and comprises 22% MPEG 5000 and 0.2 M ammonium sulfate.
3. The method of claim 1 , wherein the polypeptide is incubated in the buffered solution at a temperature range of 2° C. to 22° C.
4. The method of claim 1 , wherein the polypeptide is incubated in the buffered solution at a temperature of 22° C.
5. The method of claim 1 , wherein the polypeptide is incubated in the buffered solution from 2 to 30 days.