Renaturation of casein kinase ii from recombinant subunits produced in escherichia coli: Purification and characterization of the reconstituted holoenzyme

Wey Jinq Lin, Jolinda A. Traugh

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40 Scopus citations

Abstract

Casein kinase II is a heterotetrameric protein kinase with an α2β2 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°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 α (catalytic) and β (regulatory) subunits of casein kinase II from Drosophila were cloned and overexpressed with the pET3a vector in Eseherichia 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 a subunit was solubilized and renatured in an approximate 1:1 ratio with the β subunit, the catalytic activity was stimulated fourfold over that of the α 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 α2β2 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 KC1 concentration required for optimal activity. The conditions described herein for the reconstitution of casein kinase II will enable investigation of the enzymatic properties of casein kinase II through recombinant DNA techniques.

Original languageEnglish
Pages (from-to)256-264
Number of pages9
JournalProtein Expression and Purification
Volume4
Issue number3
DOIs
StatePublished - Jun 1993

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