Over-expression and purification of isotopically labeled recombinant ligand-binding domain of orphan nuclear receptor human B1-binding factor/human liver receptor homologue 1 for NMR studies.
The human hepatitis B virus enhancer II B1 binding factor (hB1F), which regulates the expression of hepatitis B virus genes, is identified as a nuclear receptor. It regulates several liver-specific genes and plays an important role in the bile acid biosynthesis pathway. A significantly optimized protocol has been worked out to prepare 15N and/or 13C-labeled hB1F ligand-binding domain in minimal medium with high yields for NMR studies. Under the various conditions optimized for the purification of His6-hB1F ligand-binding domain, the yield of the purified protein is estimated to be 25-30 mg from 0.5 L of M9 minimal media. Electrospray ionization mass spectrometry data confirm the correctness of the primary sequence. Dynamic light scattering experiment proves that the protein exists as a monomeric form. In addition, the circular dichroism results show that the protein has a well-regulated secondary structure and a high alpha-helical content in ammonium bicarbonate buffer at 20 degrees C and pH 7.4. Finally, uniformly 15N-labeled protein is characterized by a TROSY-HSQC spectrum, and the dispersion of 15N-1H cross-peaks in the spectrum indicates the presence of well-ordered and properly folded protein as a monomer.
Chen
Xiang
X
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.
Tong
Xiaotian
X
Xie
Youhua
Y
Wang
Yuan
Y
Ma
Jinbiao
J
Gao
DaMing
D
Wu
Houming
H
Chen
Haibao
H
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008024:Ligands; D008027:Light; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012542:Scattering, Radiation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D008024:Ligands; C082631:NR5A2 protein, human; D009587:Nitrogen Isotopes; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors