Expression and purification of human recombinant GST-FGF receptor-1.
When tumors undergo the angiogenic switch, cell growth and tissue invasion is facilitated by the formation of new capillaries from preexisting blood vessels, a process known as angiogenesis. Growth factors such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and fibroblast growth factor (FGF) trigger the process of angiogenesis. Here we describe a protocol for the expression and one-step purification of human recombinant GST-FGF receptor type 1 (FGFR-1) from Sf9 cells. This protocol allows generating an active kinase as indicated by its reactivity with a monoclonal antibody to phosphorylated tyrosine. The purified enzyme displays a specific activity of 1.2 x 10(4) pmol mg(-1) min(-1), which is in the range of activities reported for homogeneously purified recombinant kinases. We have employed a number of compounds to show that the GST-FGFR-1 preparation is suitable to the identification of tyrosine kinase inhibitors. Considering that inhibitors of angiogenesis may represent an attractive tool in therapeutic strategies targeting invasive metastatic tumors the results presented here, along with available data on the structure of the ATP-binding pocket of FGFR-1, should facilitate the rational design of specific FGFR-1 inhibitory compounds.
Homann
S
S
Department of Oncology, Novartis Pharma AG, K-125.4.01, Klybeckstrasse 141, CH-4057, Basel, Switzerland.
Schacher
B
B
Zumstein-Mecker
S
S
Fabbro
D
D
Bold
G
G
Ferrari
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D016220:Fibroblast Growth Factor 1; D005615:Freezing; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D016220:Fibroblast Growth Factor 1; D019000:Phosphotyrosine; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; C496348:FGFR1 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1