Expression of stable hepatitis B viral polymerase associated with GRP94 in E. coli.
We here presented evidence that a 94-kDa glucose-regulated protein (GRP94) was associated with hepatitis B viral (HBV) polymerase in the human liver cell HepG2 and this association could be applied even in Escherichia coli. We investigated the role of GRP94 in the expression and stabilization of HBV polymerase in Escherichia coli by coexpression of the two proteins. The affinity column-purified glutathione S-transferase-tagged HBV polymerase (GST-P, 130 kDa) showed a proper molecular size and reverse transcriptase activity on several exogenous templates and was sensitive to specific inhibitors. The GST-P was associated with the maltose-binding protein-tagged GRP94 (MBP-GRP94, 130 kDa) using analyses by an affinity chromatography, native gel electrophoresis and glycerol gradient centrifugation. However, nondenaturing and partially denaturing activity gel analyses showed two active bands of approximately 260 kDa and approximately 130 kDa, respectively. Furthermore, in the presence of the encapsidation signal RNA template (HBV epsilon RNA), the approximately 260-kDa active band was gradually converted to approximately 130 kDa, which implies that HBV polymerase was dissociated from the chaperone GRP94 and bound preferentially to the HBV epsilon RNA. These results suggested that the chaperone GRP94 was necessary for the stabilization and production of HBV polymerase as an active form.
Kim
S S
SS
School of Biological Sciences, Seoul National University, Korea.
Shin
H J
HJ
Cho
Y H
YH
Rho
H M
HM
D002460:Cell Line; D004795:Enzyme Stability; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral; D015684:Gene Products, pol / Q000096:biosynthesis* / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006509:Hepatitis B / Q000378:metabolism / Q000821:virology*; D006515:Hepatitis B virus / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D015684:Gene Products, pol; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; C066027:P protein, Hepatitis B virus; C018551:glucose-regulated proteins; D012194:RNA-Directed DNA Polymerase