Expression and purification of recombinant human fibroblast growth factor receptor in Escherichia coli.
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.
Ryu
Eui Kyung
EK
Department of Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
Cho
Ki Joon
KJ
Kim
Jin Kwang
JK
Harmer
Nicholas J
NJ
Blundell
Tom L
TL
Kim
Kyung Hyun
KH
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
D018832:Molecular Chaperones; D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1; D051496:Receptor, Fibroblast Growth Factor, Type 1