title: Simultaneous protein expression and modification: an efficient approach for production of unphosphorylated and biotinylated receptor tyrosine kinases by triple infection in the baculovirus expression system.
abstract: Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin-biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and site-specific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for enzymatic and biophysical studies.
authors.affiliation: Druggability Enzymology Profiling Unit, Novartis Institutes for BioMedical Research, Oncology, Novartis Pharma AG, Basel, Switzerland. dirk.erdmann@novartis.com
authors.forename: Dirk
authors.initials: D
authors.lastname: Erdmann
authors.forename: Catherine
authors.initials: C
authors.lastname: Zimmermann
authors.forename: Patrizia
authors.initials: P
authors.lastname: Fontana
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Hau
authors.forename: Alain
authors.initials: A
authors.lastname: De Pover
authors.forename: Patrick
authors.initials: P
authors.lastname: Chène
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D015153:Blotting, Western; D002457:Cell Extracts; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D008967:Molecular Biology / Q000379:methods*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D002457:Cell Extracts; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin