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J Biol Chem, Vol. 273, Issue 29, 18060-18066, July 17, 1998

Rapid, High Level Protein Production Using DNA-based Semliki Forest Virus Vectors

David P. DiCiommoDagger § and Rod BremnerDagger parallel

From the Dagger  Eye Research Institute of Canada, Toronto, Ontario M5T 2S8 and the § Department of Medical Genetics and Microbiology and parallel  Departments of Ophthalmology and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada

Semliki Forest virus (SFV) vectors can be produced faster, and have a wider host range, than baculovirus vectors. However, the original SFV system requires in vitro manipulation of RNA. We have generated a system that is wholly DNA-based. Both the replicon vector, encoding SFV polymerase and the protein of interest, and the helper vector, encoding viral structural proteins, were modified so that expression was RNA polymerase II-dependent. Transfection of the modified replicon plasmid alone generated 20-30-fold more protein than obtained from a simple expression vector. Expression required the SFV replicase, which amplifies replicon RNA. The SFV-based vector generated 10-20-fold more protein than a plasmid based on Sindbis virus. Cotransfection of SFV replicon and helper vectors generated viral titers of around 106 infectious particles/ml. A single electroporation, plated on one 10-cm plate, generated enough virus (107 particles) to produce >500 µg of protein. Wild type, replication proficient virus was not detected in three tests utilizing almost 108 viral particles, a distinct advantage over a DNA Sindbis-based system in which over half the virus particles generated are fully infectious. The new SFV vectors significantly enhance the utility of this expression system.

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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