Isolation and molecular characterization of hepatitis B virus X-protein from a baculovirus expression system.
The X protein (HBx) of the human Hepatitis B Virus (HBV) is a regulatory protein that exercises a transcriptional activator function on a variety of regulatory elements and is therefore considered to be involved in the development of human hepatocellular carcinoma (HCC). So far, most attempts at elucidating HBx function have been undertaken at the genetic level, reflecting the difficulties in detecting the very low amounts of the protein in infected livers. Consequently, the questions of intracellular localization and posttranslational modification have not yet been completely answered. We therefore constructed recombinant baculoviruses that allowed expression of HBx and the hexa histidine HBx fusion protein HBxHis in insect cells. Cell fractionation experiments revealed that only a minor part of HBx is detectable in a soluble form in the cytosolic fraction, whereas most of the protein forms intracellular aggregates. These results could be confirmed by confocal laser immunofluorescence. The fusion of a hexa-histidine tag to the amino terminus of HBx allowed a rapid one-step purification by metal chelate affinity chromatography. The detailed analysis of purified HBxHis using electrospray ionization mass spectrometry uncovered two major components: the unmodified, monomeric, fully oxidized form with five intramolecular disulfide bridges, and its N-acetylated modification. Additionally, two minor peaks with mass differences of delta m = +80 da suggested that a small fraction of HBx becomes posttranslationally phosphorylated in insect cells. No further modifications could be observed, indicating that only phosphorylation might play a role in a possible posttranslational regulation of this viral activator.
Urban
S
S
Department of Virus Research, Max-Planck-Institut für Biochemie, München, Germany.
Hildt
E
E
Eckerskorn
C
C
Sirma
H
H
Kekulé
A
A
Hofschneider
P H
PH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D019084:Fluorescent Antibody Technique, Indirect; D013058:Mass Spectrometry; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D018411:Spodoptera; D015534:Trans-Activators / Q000737:chemistry / Q000302:isolation & purification*; D054334:Viral Regulatory and Accessory Proteins
D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein