Optimized expression from a synthetic gene of an untagged RNase H domain of human hepatitis B virus polymerase which is enzymatically active.
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.
Potenza
Nicoletta
N
Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy.
Salvatore
Vincenzo
V
Raimondo
Domenico
D
Falanga
Davide
D
Nobile
Valentina
V
Peterson
Darrel L
DL
Russo
Aniello
A
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
D017361:Viral Nonstructural Proteins; D016914:Ribonuclease H