Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.
The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays.
Savopoulos
John W
JW
GlaxoSmithKline, New Frontiers Science Park (North), Coldharbour Road, Harlow, Essex CM19 5AD, United Kingdom.
Dowd
Stephen
S
Armour
Carolyn
C
Carter
Paul S
PS
Greenwood
Catherine J
CJ
Mills
David
D
Powell
David
D
Pettman
Gary R
GR
Jenkins
Owen
O
Walsh
Frank S
FS
Philpott
Karen L
KL
D000595:Amino Acid Sequence; D016367:Baculoviridae; D019921:Biotinylation; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D015870:Gene Expression; D048056:Mitogen-Activated Protein Kinase 9; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D048056:Mitogen-Activated Protein Kinase 9; D020928:Mitogen-Activated Protein Kinases