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 Yarrowia lipolytica has been cloned and overexpressed using the pT7-7 expression 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 homogeneity. 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 renaturation by dialysis. Treatment of the enzyme found in the inclusion bodies with different nondetergent sulfobetaines (NDSB) led to a time-dependent activation. NDSB195 is a V-type activator of the recombinant catalytic subunit of casein kinase II. The NDSB195-activated enzyme remained active at the room temperature for weeks. Kinetic properties of the recombinant casein kinase II subunit are similar to those of the purified holoenzyme (low Km for ATP and inhibition by heparin). Kinetic study indicates that the beta subunit could interact with the alpha subunit at the level of the catalytic site to enhance activity and to modify the kinetic behavior of the enzyme.
Laboratoire de Chimie Biologique INRA INA-PG, Centre de Biotechnologie Agro-Industrielle, Thiverval-Grignon, 78850, France.
P H
PH
Benetti
S I
SI
Kim
D
D
Chaillot
M
M
Canonge
T
T
Chardot
J C
JC
Meunier
D001483:Base Sequence; D047390:Casein Kinase II; D002384:Catalysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology*
D017931:DNA Primers; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases