title: Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells.
abstract: The family of fibroblast growth factor receptors (FGFRs) plays an important role in cell growth, survival, differentiation and angiogenesis. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) are critical for ligand binding and specificity towards fibroblast growth factor and heparan sulfate. Fibroblast growth factor receptors are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we have cloned, expressed in CHO cells and purified Fc-fused extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2b-Fc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGFR4b-Fc), which could be used as molecular targets for the selection of specific antibodies. The fusion proteins were analyzed using gel electrophoresis, Western blotting and mass spectrometry. To facilitate their full characterization, the fusion proteins were deglycosylated using PNGase F enzyme. With an optimized transient transfection protocol and purification procedure we were able to express the proteins at a high level and purify them to homogeneity.
authors.affiliation: Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Sokolowska-Wedzina
authors.affiliation: Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Borek
authors.affiliation: Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland.
authors.forename: Julia
authors.initials: J
authors.lastname: Chudzian
authors.affiliation: Faculty of Biotechnology, Department of Protein Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Jakimowicz
authors.affiliation: Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Zakrzewska
authors.affiliation: Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland. Electronic address: otlewski@protein.pl.
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000302:isolation & purification*; D007074:Immunoglobulin G; D013058:Mass Spectrometry; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins
keywords: CHO; FGFR; Glycosylation; Protein expression