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 N-15 and/or C-13-labeled hB1F ligand-binding domain in minimal medium with high yields for NMR studies. Under the various conditions optimized for the purification of His(6)-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 N-15-labeled protein is characterized by a TROSY-HSQC spectrum, and the dispersion of N-15-H-1 cross-peaks in the spectrum indicates the presence of well-ordered and properly folded protein as a monomer. (C) 2005 Elsevier Inc. All rights reserved.