Renaturation of casein kinase II from recombinant subunits produced in Escherichia coli: purification and characterization of the reconstituted holoenzyme.
Casein kinase II is a heterotetrameric protein kinase with an alpha 2 beta 2 composition; the subunits can be separated only under harsh denaturing conditions. In this study, the optimal conditions for renaturation of denatured casein kinase II have been established. Purified casein kinase II from rabbit reticulocytes was denatured with 8 M urea and 0.1 M dithiothreitol at 25 degrees C. Various parameters, including arginine, oxidized glutathione/dithiothreitol, substrate, and temperature were optimized for renaturation. Under optimal conditions, the denatured protein kinase was successfully renatured with a recovery of 75% activity and eluted around 160,000 Da upon gel filtration, indicating the tetrameric structure. When the alpha (catalytic) and beta (regulatory) subunits of casein kinase II from Drosophila were cloned and overexpressed with the pET3a vector in Escherichia coli, the majority of the subunits were in an insoluble form. The optimal conditions for denaturation/renaturation of the recombinant casein kinase II from Drosophila were identical to those developed for the holoenzyme, except for the redox state and temperature. When the alpha subunit was solubilized and renatured in an approximate 1:1 ratio with the beta subunit, the catalytic activity was stimulated fourfold over that of the alpha subunit alone. The reconstituted enzyme was purified to apparent homogeneity in one step by chromatography on heparin--TSK. Gel filtration indicated the formation of an alpha 2 beta 2 tetramer. The reconstituted recombinant casein kinase II exhibited characteristics of the native holoenzyme in subunit composition, inhibition by heparin, stimulation by basic compounds, and the KCl concentration required for optimal activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Lin
W J
WJ
Department of Biochemistry, University of California, Riverside 92521.
Traugh
J A
JA
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D047390:Casein Kinase II; D003001:Cloning, Molecular; D004330:Drosophila / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D002479:Inclusion Bodies / Q000201:enzymology; D011489:Protein Denaturation; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014508:Urea; D001120:Arginine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases