Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain.
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.
Razzera
Guilherme
G
Laboratório de Expressão Gênica, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil.
Vernal
Javier
J
Portugal
Rodrigo V
RV
Calgaro
Marcos R
MR
Fernandez
Pablo
P
Zakin
Mario M
MM
Polikarpov
Igor
I
Terenzi
Hernán
H
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
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