Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain
| Autor: | Mario M. Zakin, Marcos R. Calgaro, Igor Polikarpov, Pablo Fernandez, Hernán Terenzi, Guilherme Razzera, Rodrigo Villares Portugal, Javier Vernal |
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| Rok vydání: | 2004 |
| Předmět: |
Protein Folding
Receptors Steroid Time Factors Protein Conformation Ultraviolet Rays Genetic Vectors Molecular Sequence Data Receptors Cytoplasmic and Nuclear Biology Ligands Mass Spectrometry Transactivation Protein structure Sequence Analysis Protein Escherichia coli Nuclear Receptor Subfamily 4 Group A Member 1 Animals Amino Acid Sequence Binding site Receptor Binding Sites Circular Dichroism Ligand (biochemistry) Recombinant Proteins Protein Structure Tertiary Rats Neuron-derived orphan receptor 1 DNA-Binding Proteins Biochemistry Nuclear receptor ROR1 Electrophoresis Polyacrylamide Gel Protein Binding Transcription Factors Biotechnology |
| Zdroj: | Protein Expression and Purification. 37:443-449 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2004.06.024 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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