Preparation of large-volume crystals for structure analysis of human casein kinase-2 by neutron crystallography

Shibazaki, Chie; Adachi, Motoyasu; Hiromoto, Takeshi; Shimizu, Rumi; Kuroki, Ryota

Casein kinase 2 (CK2) is one of the ubiquitous Ser/Thr kinases and is involved in the cell cycle and the survival and proliferation of cells. CK2 is a heterotetrameric structure comprising two - or -subunits and two regulatory -subunits. In order to understand the biological function of the alpha catalytic subunit of CK2, we aim to analyze the structure of CK2 including information of the hydrogen and hydrating water molecule by neutron crystallography. The gene coding CK2 was inserted into pET24a and expressed in E. coli strain BL21DE3, in which the mobile region and chemically reactive thiols were removed by amino acid mutation. A total of 150 mg protein was obtained from a 6 L culture, and was used for crystallization trials. The preparation of large crystals was performed using a macro seeding method specially developed for CK2. Finally, a large crystal with a volume of approximately 2 mm was reproducibly obtained. From the X-ray diffraction study, we confirmed that the crystals obtained diffracted to approx. 1 resolution at 100 K after soaking the crystal into the deuterated cryo protectant. The neutron diffraction data collection is planned to obtain a high resolution neutron structure of CK2.