title: Optimized expression from a synthetic gene of an untagged RNase H domain of human hepatitis B virus polymerase which is enzymatically active.
abstract: The RNase H domain of human hepatitis B virus (HBV) polymerase is an attractive molecular target for the development of new anti-HBV drugs. In this study, a synthetic gene coding for HBV RNase H was assembled from 12 oligonucleotides and expressed in Escherichia coli. The encoded protein was then recovered from inclusion bodies, purified, and refolded by a dilution-dialysis procedure in the presence of a low concentration of lauroylsarcosine (0.01%). The presence of the detergent was an absolute requirement for solubility, suggesting that the untagged RNase H might have exposed hydrophobic regions that need to be shielded from the solvent. The structural identity of the protein was confirmed by N-terminal amino acid sequence analysis and mass spectrometry. The enzymatic activity of HBV RNase H was then tested by a recently developed fluorometric assay and was found to be only slightly lower than that registered with the entire HIV-1 reverse transcriptase. Finally, a structural model of the enzyme showed that H715, R744 and K745 may be involved in substrate recognition.
authors.affiliation: Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Potenza
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Salvatore
authors.forename: Domenico
authors.initials: D
authors.lastname: Raimondo
authors.forename: Davide
authors.initials: D
authors.lastname: Falanga
authors.forename: Valentina
authors.initials: V
authors.lastname: Nobile
authors.forename: Darrel L
authors.initials: DL
authors.lastname: Peterson
authors.forename: Aniello
authors.initials: A
authors.lastname: Russo
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000201:enzymology*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016914:Ribonuclease H / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D017361:Viral Nonstructural Proteins; D016914:Ribonuclease H