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Functional imaging of proteins in the MAP kinase signaling cascade


Vuckovic,  M.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Vuckovic, M. (2004). Functional imaging of proteins in the MAP kinase signaling cascade. Diploma Thesis, Georg-August-Universität, Göttingen, Germany.

Cite as: http://hdl.handle.net/11858/00-001M-0000-0012-EDEE-7
It has been reported that recombinant proteins containing tetracysteine tags can be fluorescently labeled in living cells. The membrane-permeant fluorescein derivative, FlAsH-EDT₂, 4’,5’-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ehtanedithiol)₂, exhibits green fluorescence only upon binding with high affinity to a specific tetracysteine motif (CCXXCC, where XX are optimized to PG). Various analogues of FlAsH have been synthesized, including ReAsH, a red fluorescing resorufin derivative. The aim of the study was to use FlAsH-EDT₂ and ReAsH-EDT₂ for site specific labeling of human extracellular regulated kinase 1 (herk1), a member of the MAP Kinase signaling cascade. For this purpose, the recombinant human ERK1 with a FlAsH binding domain (FBD) DCCPGCCK at the C-terminus was designed. hERK1-FBD was transiently transfected in mouse embryonic fibroblasts (MEF), which are erk1 null, and HeLa cells. In addition, FlAsH binding to endogenous cellular proteins that have sequence similarity to the FlAsH target CCPGCC was tested in vitro.