Rapid, high level protein production using DNA-based Semliki Forest virus vectors.
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 10(6) infectious particles/ml. A single electroporation, plated on one 10-cm plate, generated enough virus (10(7) particles) to produce >500 microg of protein. Wild type, replication proficient virus was not detected in three tests utilizing almost 10(8) 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.
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Eye Research Institute of Canada, Toronto, Ontario M5T 2S8.
Bremner
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D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D019556:COS Cells; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D017930:Genes, Reporter; D005822:Genetic Vectors; D017874:Immediate-Early Proteins / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012093:Replicon / Q000235:genetics; D012672:Semliki forest virus / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics
D000956:Antigens, Viral; D004279:DNA, Viral; D017874:Immediate-Early Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; C044582:immediate-early proteins, cytomegalovirus; C070852:RNA polymerase SP6; D012321:DNA-Directed RNA Polymerases