title: Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system.
abstract: Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.
authors.affiliation: Protein Sciences, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA. Electronic address: kelly.yan@novartis.com.
authors.forename: Kelly
authors.initials: K
authors.lastname: Yan
authors.affiliation: Biochemical Lead Discovery, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA.
authors.forename: Hanne
authors.initials: H
authors.lastname: Merritt
authors.affiliation: Protein Sciences, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Crawford
authors.affiliation: Protein Sciences, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA.
authors.forename: Gwynn
authors.initials: G
authors.lastname: Pardee
authors.affiliation: Protein Sciences, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA.
authors.forename: Jan Marie
authors.initials: JM
authors.lastname: Cheng
authors.affiliation: Protein Sciences, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA.
authors.forename: Stephania
authors.initials: S
authors.lastname: Widger
authors.affiliation: Biochemical Lead Discovery, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Hekmat-Nejad
authors.affiliation: Protein Sciences, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA.
authors.forename: Isabel
authors.initials: I
authors.lastname: Zaror
authors.affiliation: Protein Sciences, Novartis Institutes for BioMedical Research, Inc., 5300 Chiron Way, Emeryville, CA 94608, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Sim
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human
keywords: Anion exchange chromatography; Biochemical assay; Co-expression; ERK2 (extracellular signal-regulated kinase 2); Insect expression; Phosphorylation