Overexpression, Purification, and Crystal Structure of Native ERα LBD

Several crystal structures of human estrogen receptor α ligand-binding domain (hERα LBD) complexed with agonist or antagonist molecules have previously been solved. The proteins had been modified in cysteine residues (carboxymethylation) or renatured in urea to circumvent aggregation and denaturatio...

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Bibliographic Details
Published in:Protein expression and purification 2001-07, Vol.22 (2), p.165-173
Main Authors: Eiler, Sylvia, Gangloff, Monique, Duclaud, Sylvie, Moras, Dino, Ruff, Marc
Format: Article
Language:English
Subjects:
Cloning, Molecular
Computer Simulation
crystal structure
Crystallization
Crystallography, X-Ray
Dimerization
estradiol
Estrogen Receptor alpha
expression
Humans
Ligands
Models, Molecular
nuclear receptor
Peptide Fragments - chemistry
Peptide Fragments - genetics
Peptide Fragments - isolation & purification
Peptide Fragments - metabolism
Protein Folding
Protein Structure, Secondary - genetics
Protein Structure, Tertiary - genetics
purification
Receptors, Estrogen - chemistry
Receptors, Estrogen - genetics
Receptors, Estrogen - isolation & purification
Receptors, Estrogen - metabolism
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - isolation & purification
Recombinant Fusion Proteins - metabolism
steroid
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Description
Summary:Several crystal structures of human estrogen receptor α ligand-binding domain (hERα LBD) complexed with agonist or antagonist molecules have previously been solved. The proteins had been modified in cysteine residues (carboxymethylation) or renatured in urea to circumvent aggregation and denaturation problems. In this work, high-level protein expression and purification together with crystallization screening procedure yielded high amounts of soluble protein without renaturation or modifications steps. The native protein crystallizes in the space group P32 21 with three molecules in the asymmetric unit. The overall structure is very similar to that previously reported for the hERα LBD with cysteine carboxymethylated residues thus validating the modification approach. The present strategy can be adapted to other cases where the solubility and the proper folding is a difficulty.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.2001.1409