Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain

Autor(es)

Razzera, Guilherme ; Vernal, Javier ; Portugal, Rodrigo V. ; Calgaro, Marcos R. ^[4] ; Fernandez, Pablo ; Zakin, Mario M. ; Polikarpov, Igor ; Terenzi, Hernán

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 37, n. 2, p. 443-449, Oct. 2004.
• Área do conhecimento: Ciências Biológicas - Biofísica
• Assunto(s): Biofísica Receptores citoplasmáticos e nucleares Bactérias Escherichia coli

Resumo

NOR-1 is an orphan member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, fatty acids, retinoic acids, and other lipophilic molecules. The NOR-1 subfamily (NR4), composed also of Nurr1 and Nurr77, has been implicated in cell proliferation, differentiation, apoptosis, chondrosarcomas, inflammation, and atherogenesis. The NOR-1 receptor is an orphan ligand receptor which acts over gene transactivation. No ligands, if such in fact exist, are known for this receptor. Recently, the three-dimensional structure of the homolog receptor Nurr1 has been solved using protein crystallography techniques. Surprisingly, the structure does not present either a typical cavity for ligand binding or a classical co-factor binding site in the ligand binding domain (LBD). To allow for structural studies of other members of NR4 subfamily, we have subcloned, overexpressed in Escherichia coli cells, purified, and characterized the rat orphan nuclear receptor NOR-1 LBD domain. We obtained NOR-1 LBD at a high degree of purity and with an overall yield of 3 mg/L of culture media. CD spectroscopic analysis shows a high α-helical secondary structure content (52%), similar to that of Nurr 1 LBD three-dimensional structure. Thermal denaturation monitored by UV absorption and CD spectroscopy suggests proper folding of recombinant NOR-1 LBD. (AU)

• Processo FAPESP: 99/03387-4 - Estudos estruturais de proteínas usando luz síncrotron
• Beneficiário: Igor Polikarpov
• Modalidade de apoio: Auxílio à Pesquisa - Temático