EXPRESSION AND CHARACTERIZATION OF THE RECOMBINANT CATALYTIC SUBUNIT OF CASEIN KINASE-II FROM THE YEAST YARROWIA-LIPOLYTICA IN ESCHERICHIA-COLI

The alpha catalytic subunit of casein kinase II from the yeast Yarrowi a lipolytica has been cloned and overexpressed using the pT7-7 express ion vector in Escherichia coli. Casein kinase activity is found in the bacterial extracts. The catalytic subunit is partially expressed in a soluble and active form, which is purified to electrophoretic homogen eity. Most of the enzyme was found in inclusion bodies. In this form, the enzyme, which is almost pure, exhibits a low specific activity. We have focused our efforts on methods to activate the protein from the inclusion bodies. We have studied the renaturation of urea-denaturated CKII catalytic subunit. We have tried different renaturation buffers and found that renaturation by dilution was more efficient than renatu ration by dialysis. Treatment of the enzyme found in the inclusion bod ies with different nondetergent sulfobetaines (NDSB) led to a time-dep endent activation. NDSB195 is a V-type activator of the recombinant ca talytic subunit of casein kinase II. The NDSB195-activated enzyme rema ined active at the room temperature for weeks. Kinetic properties of t he recombinant casein kinase II subunit are similar to those of the pu rified holoenzyme (low K-m for ATP and inhibition by heparin). Kinetic study indicates that the beta subunit could interact with the alpha s ubunit at the level of the catalytic site to enhance activity and to m odify the kinetic behavior of the enzyme. (C) 1998 Academic Press.