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Expression, Purification and Characterization of Inactive and Active Forms of ERK2 from Insect Expression System

Yan, Kelly and Merritt, Hanne and Crawford, Kenneth and Pardee, Gwynn and Cheng, Jan Marie and Widger, Stephania and Hekmat-Nejad, Mohammad and Zaror, Isabel and Sim, Janet (2015) Expression, Purification and Characterization of Inactive and Active Forms of ERK2 from Insect Expression System. Protein Expression and Purification.

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.

Item Type: Article
Keywords: ERK2 (extracellular signal-regulated kinase 2), insect expression, co-expression, phosphorylation, anion exchange chromatography, biochemical assay
Date Deposited: 28 Apr 2016 23:45
Last Modified: 28 Apr 2016 23:45
URI: https://oak.novartis.com/id/eprint/24824

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