Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies.
The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non-small-cell lung cancer). Small-molecule-based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells).1H,15N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well-folded but unstable, and it did not crystallize. However, a construct (D596-G900) produced in Sf9 cells yielded homogenous, well-folded protein that crystallized readily, thereby resulting in eleven new EPHA2-ligand crystal structures. We have also established a strategy for selective and uniform15N-amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2-drug interactions by NMR.
Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-Universität, Max-von-Laue-Strasse 7, 60438, Frankfurt am Main, Germany.|German Cancer Consortium (DKTK), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.|German Cancer Research Center (DKFZ), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Santosh L
SL
Gande
Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-Universität, Max-von-Laue-Strasse 7, 60438, Frankfurt am Main, Germany.|German Cancer Consortium (DKTK), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.|German Cancer Research Center (DKFZ), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Krishna
K
Saxena
Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-Universität, Max-von-Laue-Strasse 7, 60438, Frankfurt am Main, Germany.
Sridhar
S
Sreeramulu
Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-Universität, Max-von-Laue-Strasse 7, 60438, Frankfurt am Main, Germany.
Verena
V
Linhard
Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-Universität, Max-von-Laue-Strasse 7, 60438, Frankfurt am Main, Germany.|German Cancer Consortium (DKTK), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.|German Cancer Research Center (DKFZ), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Denis
D
Kudlinzki
German Cancer Consortium (DKTK), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.|German Cancer Research Center (DKFZ), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.|Chair of Proteomics and Bioanalytics, Technical University of Munich, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.
Stephanie
S
Heinzlmeir
Chair of Biological Chemistry, Technical University of Munich, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.
Andreas J
AJ
Reichert
Chair of Biological Chemistry, Technical University of Munich, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.
Arne
A
Skerra
German Cancer Consortium (DKTK), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.|German Cancer Research Center (DKFZ), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.|Chair of Proteomics and Bioanalytics, Technical University of Munich, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.|Center for integrated Protein Science Munich (CIPSM), Technical University of Munich, Arcisstrasse 21, 80333, München, Germany.|Bavarian Biomolecular Mass Spectrometry Center, Technical University of Munich, Gregor-Mendel-Strasse 4, 85354, Freising, Germany.
Bernhard
B
Kuster
Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-Universität, Max-von-Laue-Strasse 7, 60438, Frankfurt am Main, Germany.|German Cancer Consortium (DKTK), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.|German Cancer Research Center (DKFZ), In Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Harald
H
Schwalbe
D000818:Animals; D005591:Chemical Fractionation; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D000072417:Protein Domains; D036104:Receptor, EphA2 / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism
D036104:Receptor, EphA2
EPHA2; NMR spectroscopy; crystallography; kinase; protein expression; structural biology