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 v essels, a process known as angiogenesis. Growth factors such as vascular en dothelial growth factor/vascular permeability factor (VEGF/VPF) and fibrobl ast growth factor (FGF) trigger the process of angiogenesis. Were we descri be a protocol for the expression and one-step purification of human recombi nant GST-FGF receptor type 1 (FGFR-1) from Sf9 cells. This protocol allows generating an active kinase as indicated by its reactivity with a monoclona l antibody to phosphorylated tyrosine. The purified enzyme displays a speci fic activity of 1.2 x 10(4) pmol mg(-1) min(-1), which is in the range of a ctivities reported for homogeneously purified recombinant kinases. We have employed a number of compounds to show that the GST-FGFR-1 preparation is s uitable to the identification of tyrosine kinase inhibitors. Considering th at inhibitors of angiogenesis may represent an attractive tool in therapeut ic strategies targeting invasive metastatic tumors the results presented he re, along with available data on the structure of the ATP-binding pocket of FGFR-1, should facilitate the rational design of specific FGFR-1 inhibitor y compounds. (C) 2001 Elsevier Science B.V. All rights reserved.