Optimized expression from a synthetic gene of an untagged RNase H domain of human hepatitis B virus polymerase which is enzymatically active

Nicoletta, Potenza; Vincenzo, Salvatore; Raimondo, Domenico; Davide, Falanga; Valentina, Nobile; Peterson, Darrel L.; Aniello, Russo

doi:10.1016/j.pep.2007.04.005

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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. (C) 2007 Elsevier Inc. All rights reserved.

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Titolo:	Optimized expression from a synthetic gene of an untagged RNase H domain of human hepatitis B virus polymerase which is enzymatically active
Autori:	Nicoletta Potenza Vincenzo Salvatore RAIMONDO, Domenico Davide Falanga Valentina Nobile Darrel L. Peterson Aniello Russo mostra contributor esterni nascondi contributor esterni
Data di pubblicazione:	2007
Rivista:	PROTEIN EXPRESSION AND PURIFICATION
Citazione:	Optimized expression from a synthetic gene of an untagged RNase H domain of human hepatitis B virus polymerase which is enzymatically active / Nicoletta, Potenza; Vincenzo, Salvatore; Raimondo, Domenico; Davide, Falanga; Valentina, Nobile; Darrel L., Peterson; Aniello, Russo. - In: PROTEIN EXPRESSION AND PURIFICATION. - ISSN 1046-5928. - STAMPA. - 55:1(2007), pp. 93-99.
Handle:	http://hdl.handle.net/11573/395910
Appartiene alla tipologia:	01a Articolo in rivista

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