Expression and glycosylation studies of human FGF receptor 4.
Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs. FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design. Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4(ed)) for structural studies. We show that baculovirus-insect cell-expressed FGFR4(ed) is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated. The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4(ed), but was still heterogeneous. Large amounts of FGFR4(ed) have been produced into inclusion bodies in Escherichia coli and refolded at least partly correctly but the refolded E. coli-produced FGFR4(ed) still aggregates.
Tuominen
H
H
Turku Centre for Biotechnology, University of Turku and Abo Akademi University, FIN-20521, Turku, Finland.
Heikinheimo
P
P
Loo
B M
BM
Kataja
K
K
Oker-Blom
C
C
Uutela
M
M
Jalkanen
M
M
Goldman
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D002479:Inclusion Bodies / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D020673:Protein Renaturation; D051499:Receptor, Fibroblast Growth Factor, Type 4; D017468:Receptors, Fibroblast Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D006493:Heparin; C496371:FGFR4 protein, human; D051499:Receptor, Fibroblast Growth Factor, Type 4; D014357:Trypsin