Expression and purification of the alpha and beta subunits of Drosophila casein kinase II using a baculovirus vector.
Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.
Birnbaum
M J
MJ
Department of Biochemistry, University of Georgia, Athens 30602.
Wu
J
J
O'Reilly
D R
DR
Rivera-Marrero
C A
CA
Hanna
D E
DE
Miller
L K
LK
Glover
C V
CV
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D047390:Casein Kinase II; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D009036:Moths; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases