title: Expression and purification of recombinant human fibroblast growth factor receptor in Escherichia coli.
abstract: Human fibroblast growth factor receptor (FGFR) is responsible for multifunctional signaling that regulates developmental processes. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) include the determinants of ligand binding and specificity for fibroblast growth factor and heparan sulfate. D1 and the D1-D2 linker with a contiguous stretch of acidic amino acids are known to be involved in auto-inhibitory regulation. In an effort to gain a better understanding of the role of D1 and the linker in FGFR regulation, we have subcloned, overexpressed, and purified the extracellular fragments, D1-D2 and D1-D3, of FGFR1 in Escherichia coli. The recombinant proteins were produced in an insoluble form and were renatured using a dropwise or on-column refolding method. In addition, D2-D3 was coexpressed with chaperones to test the possibility that the presence of chaperones might enhance refolding efficiencies. A combination of immobilized nickel and heparin affinity chromatography and size-exclusion chromatography resulted in the purification of recombinant ectodomain proteins D1-D2 and D1-D3 of high purity for structural studies.
authors.affiliation: Department of Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
authors.forename: Eui Kyung
authors.initials: EK
authors.lastname: Ryu
authors.forename: Ki Joon
authors.initials: KJ
authors.lastname: Cho
authors.forename: Jin Kwang
authors.initials: JK
authors.lastname: Kim
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Harmer
authors.forename: Tom L
authors.initials: TL
authors.lastname: Blundell
authors.forename: Kyung Hyun
authors.initials: KH
authors.lastname: Kim
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018832:Molecular Chaperones; D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1; D051496:Receptor, Fibroblast Growth Factor, Type 1