Synthesis of biologically active adenovirus preterminal protein in insect cells using a baculovirus vector.
A DNA fragment encoding the polyhedrin promoter of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV strain) was constructed using overlapping oligodeoxyribonucleotides (oligos), which included the 5'-untranslated leader sequence of the polyhedrin-encoding gene. This DNA fragment was cloned into an intermediate transfer vector (pKX105) providing a unique BamHI site for the insertion of foreign genes. The Escherichia coli lacZ gene was first cloned at the BamHI site of pKX105 and the XhoI-KpnI fragment containing the lacZ gene was transferred to another plasmid vector (pEI) consisting of flanking AcMNPV sequences (pEI-lacZ). The E. coli beta-galactosidase that was produced in the infected insect cells using the recombinant virus constituted about 10% of the total cytoplasmic proteins. The pKX105 plasmid was also modified to give rise to pTT-lacZ which consisted of the lacZ gene under the control of the Rous sarcoma virus long terminal repeat promoter to facilitate rapid screening of the baculoviral recombinants in which the gene of interest was cloned under the control of the polyhedrin promoter. The efficiency of these transfer vectors was verified by obtaining high levels of expression of the adenovirus(Ad)-encoded preterminal protein (pTP) which is involved as a protein primer in the initiation of Ad DNA replication. The baculovirus-produced pTP was immunoprecipitable using rabbit polyclonal antibodies raised against a hydrophilic domain of pTP. The pTP protein was localized in the nucleus of the infected insect cells, and was biologically active in the in vitro Ad type 2 (Ad2) replication initiation assay.
Zhao
L J
LJ
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66103.
Irie
K
K
Trirawatanapong
T
T
Nakano
R
R
Nakashima
A
A
Morimatsu
M
M
Padmanabhan
R
R
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007313:Insecta; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D015183:Restriction Mapping; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D015345:Oligonucleotide Probes; D011498:Protein Precursors; D014764:Viral Proteins; C056461:terminal protein, adenovirus; D001616:beta-Galactosidase