Characterization of CK2 holoenzyme variants with regard to crystallization

A search for strategies was conducted in order to obtain a human protein ki nase CK2 preparation which would be suitable for crystallization, despite t he fact that the recombinant enzyme is abundant and can be readily purified to homogeneity. This seemingly contradiction is based on the fact that the catalytic subunit moiety of the human CK2 holoenzyme is not stable neither as a free subunit nor in the tetrameric complex. All attempts to prevent d egradation failed. Hence, alternative approaches were designed in order to avoid this degradation, which was expected to hamper any crystallization ef forts severely. One of the approaches chosen was the production of a chimer ic holoenzyme made up from a human regulatory subunit and a catalytic subun it from Z. mays. The plant catalytic subunit, in contrast to the human coun terpart is very stable and does not undergo this kind of degradation. The s econd strategy to tackle the problem of instability was to produce the homo logous recombinant human CK2 holoenzyme and then, instead of trying to avoi d degradation, attempt to accelerate degradation until all catalytic subuni t material was converted to the degraded form, i.e. a 40 kDa polypeptide.