TY - JOUR
T1 - High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus
AU - Stunnenberg, Henk G.
AU - Lange, Heidrun
AU - Philipson, Lennart
AU - van Miltenburg, Rob T.
AU - van der Vlietl, Peter C.
N1 - Funding Information:
ACKNOWLEDGEMENTS We wish to thank Dr Marshall S. Horwitz and Dr Philip R.E. Branton for gifts of anti-pol and anti-pTP antibodies, Dr. B. Zelle for pBZ9 and R.S. van Schaik for computer analysis. This study was supported in part by the Netherlands Foundation Foundation for Chemical Research (SON) with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO).
PY - 1988/3/25
Y1 - 1988/3/25
N2 - Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymaticalty active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system.
AB - Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymaticalty active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system.
UR - https://www.scopus.com/pages/publications/0024296375
U2 - 10.1093/nar/16.6.2431
DO - 10.1093/nar/16.6.2431
M3 - Article
C2 - 3362670
AN - SCOPUS:0024296375
SN - 0305-1048
VL - 16
SP - 2431
EP - 2444
JO - Nucleic acids research
JF - Nucleic acids research
IS - 6
ER -