Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector |
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Authors: | Charlotte Hauser, Heidi Fusswinkel, Jian Li, Cornelia Oellig, Reinhard Kunze, Markus Mü ller-Neumann, Manfred Heinlein, Peter Starlinger Walter Doerfler |
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Affiliation: | (1) Institute of Genetics, University of Cologne, Weyertal 121, D-5000 Cologne 41, Federal Republic of Germany;(2) Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA;(3) Present address: Department of Biotechnology, Main Laboratory, BASF Aktiengesellschaft, D-6700 Ludwigshafen/Rhein, Federal Republic of Germany |
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Abstract: | Summary The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%–2% of the newly synthesized protein. |
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Keywords: | Eukaryotic expression vector Plant gene expression in insect cells Autographa californica nuclear polyhedrosis virus Western blotting Immunofluorescence |
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