Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells.
Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.
Department of Biotechnology, Vienna Institute of BioTechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Florian
F
Krammer
Jens
J
Pontiller
Christopher
C
Tauer
Dieter
D
Palmberger
Andreas
A
Maccani
Martina
M
Baumann
Reingard
R
Grabherr
D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000502:physiology; D008156:Luciferases / Q000235:genetics; D012093:Replicon; D013696:Temperature; D014162:Transfection; D014779:Virus Replication
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases