Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERalpha in complex with synthetic ligands.
The ligand-binding domain (LBD) of human oestrogen receptor alpha was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD-ligand-coactivator peptide complexes at 2.3 A resolution. This technique is likely to be applicable to other low-solubility proteins.
Cura
Vincent
V
Laboratoire de Biologie et Génomique Structurales, IGBMC, Illkirch, France.
Gangloff
Monique
M
Eiler
Sylvia
S
Moras
Dino
D
Ruff
Marc
M
D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D020641:Polymorphism, Single Nucleotide; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D047628:Estrogen Receptor alpha; D008024:Ligands; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins