Characterization of CK2 holoenzyme variants with regard to crystallization.
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.
Guerra
B
B
Institut für Biochemie, Universität zu Köln, Germany.
Niefind
K
K
Ermakowa
I
I
Issinger
O G
OG
D000818:Animals; D002147:Calmodulin / Q000737:chemistry; D047390:Casein Kinase II; D020134:Catalytic Domain; D002417:Cattle; D002850:Chromatography, Gel; D003461:Crystallography; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007477:Ions; D008027:Light; D010766:Phosphorylation; D011107:Polylysine / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D014508:Urea / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology
D002147:Calmodulin; D007477:Ions; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011107:Polylysine; D006160:Guanosine Triphosphate; D014508:Urea; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases