Expression, purification, and characterization of an activated cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase from baculovirus-infected insect cells.
An activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase was expressed in Spodoptera frugiperda (SF9) cells from a baculovirus vector. To maximize expression and to facilitate purification of the recombinant protein, CSBP2 kinase was expressed as a carboxy-terminal fusion protein to glutathione S-transferase (GST). Under optimal conditions, 2-3 mg of GST-CSBP2 could be obtained per liter of infected cell culture. The fusion protein was easily purified from the soluble fraction of the total cell lysate under nondenaturing conditions by using a glutathione-Sepharose 4B affinity resin. As expected, the purified GST-CSBP2 fusion protein was approximately 68 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and reacted with antibodies directed toward either the GST or the CSBP amino terminus. To obtain activated CSBP2, SF9 cells were coinfected with two recombinant baculovirus vectors: one that directed the synthesis of the GST-CSBP2 fusion protein and a second vector that directed the synthesis of a constitutively active form of the CSBP activating kinase, MKK3. Coexpression of GST-CSBP2 kinase with the MKK3 activator increased GST-CSBP2 activity 8- to 10-fold based on the ability of GST-CSBP2 to phosphorylate the substrate, myelin basic protein (MBP), and the ATF2 transcription factor, in vitro. Moreover, activated GST-CSBP2 was capable of activating a bacterially derived mitogen-activated protein kinase-activating protein kinase 2 in vitro. The activity of insect-derived GST-CSBP2 was also inhibited by the CSBP inhibitor, SB202190. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of this kinase.
Cai
X Y
XY
Department of Immunology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Shanahan
M
M
Miller
K
K
Gommoll
C
C
Lundell
D
D
Zavodny
P
P
Dalie
B
B
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000138:chemical synthesis / Q000302:isolation & purification; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D003001:Cloning, Molecular; D016207:Cytokines / Q000037:antagonists & inhibitors; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D005822:Genetic Vectors / Q000138:chemical synthesis; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D048371:MAP Kinase Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases
D000894:Anti-Inflammatory Agents, Non-Steroidal; D016207:Cytokines; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; C482530:MAP2K3 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases