title: Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain.
abstract: 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 alpha-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.
authors.affiliation: Laboratório de Expressão Gênica, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil.
authors.forename: Guilherme
authors.initials: G
authors.lastname: Razzera
authors.forename: Javier
authors.initials: J
authors.lastname: Vernal
authors.forename: Rodrigo V
authors.initials: RV
authors.lastname: Portugal
authors.forename: Marcos R
authors.initials: MR
authors.lastname: Calgaro
authors.forename: Pablo
authors.initials: P
authors.lastname: Fernandez
authors.forename: Mario M
authors.initials: MM
authors.lastname: Zakin
authors.forename: Igor
authors.initials: I
authors.lastname: Polikarpov
authors.forename: Hernán
authors.initials: H
authors.lastname: Terenzi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D011987:Receptors, Steroid / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D014466:Ultraviolet Rays
chemical_list: D004268:DNA-Binding Proteins; D008024:Ligands; C539820:Nr4a1 protein, rat; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D014157:Transcription Factors