Production of active recombinant mitogen-activated protein kinases through transient transfection of 293T cells.
Mitogen-activated protein (MAP) kinases are a family of serine/threonine protein kinases that play an important role in a myriad of cellular processes, including cell proliferation, differentiation, and apoptosis. Abnormal activation of MAP kinases has been shown to participate in a variety of human diseases which include cancer, septic shock, rheumatoid arthritis, diabetes, and cardiovascular diseases. Active MAP kinase enzymes are not only valuable for basic biomedical research but are also critical for the development of pharmacological inhibitors as therapeutic drugs in the treatment of relevant human diseases. MAP kinases produced in a bacterial system are poorly active due to a lack of proper phosphorylation at their characteristic threonine and tyrosine residues. To overcome these limitations, we have developed a mammalian expression system for high level expression and one-step purification of enzymatically MAP kinases. We cloned JNK1, p38, and p38-regulated MAP kinase-activated protein kinase-2 into the mammalian expression vector pEBG, and expressed these protein kinases as glutathione S-transferase fusion proteins in human embryonic kidney 293T cells through transient transfection. The protein kinases were activated in vivo through treating the transfected cells with sodium arsenite and affinity-purified using glutathione-Sepharose beads. The enzymatic activities of these protein kinases were demonstrated by Western blot analysis and in vitro kinase assays. Our results indicate that this system is an extremely powerful tool for generating valuable reagents, and could be very valuable for proteomic studies.
Zhao
Qun
Q
Children's Research Institute, Children's Hospital, Department of Pediatrics, The Ohio State University, 700 Children's Drive, Columbus, OH 43205, USA.
Chen
Peili
P
Manson
Mary E
ME
Liu
Yusen
Y
D017209:Apoptosis / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000096:biosynthesis* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*; D014162:Transfection / Q000379:methods
D011993:Recombinant Fusion Proteins; D020929:Mitogen-Activated Protein Kinase Kinases