High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

Abstract:

: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 enzymatically 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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Stunnenberg HG,Lange H,Philipson L,van Miltenburg RT,van der Vliet PC

doi

10.1093/nar/16.6.2431

subject

Has Abstract

pub_date

1988-03-25 00:00:00

pages

2431-44

issue

6

eissn

0305-1048

issn

1362-4962

journal_volume

16

pub_type

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