Journal
MOLECULAR AND CELLULAR BIOCHEMISTRY
Volume 227, Issue 1-2, Pages 3-11Publisher
KLUWER ACADEMIC PUBL
DOI: 10.1023/A:1013184000557
Keywords
CK2; crystallization; truncated protein kinases; protein purification
Categories
Ask authors/readers for more resources
A search for strategies was conducted in order to obtain a human protein kinase CK2 preparation which would be suitable for crystallization, despite the 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 degradation failed. Hence, alternative approaches were designed in order to avoid this degradation, which was expected to hamper any crystallization efforts severely. One of the approaches chosen was the production of a chimeric holoenzyme made up from a human regulatory subunit and a catalytic subunit from Z. mays. The plant catalytic subunit, in contrast to the human counterpart is very stable and does not undergo this kind of degradation. The second strategy to tackle the problem of instability was to produce the homologous recombinant human CK2 holoenzyme and then, instead of trying to avoid degradation, attempt to accelerate degradation until all catalytic subunit material was converted to the degraded form, i.e. a 40 kDa polypeptide.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available