Abstract
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.
Highlights
Expression of therapeutic proteins in mammalian cells is a growing field in biotechnology
Transfection efficiency The efficiency of transfecting mammalian cells can be influenced by a number of factors like the choice of method, condition of the cells, purity of DNA, plasmid size and number of different ones to be delivered per cell
The influenza replicon that we have chosen to evaluate is based on two plasmids (17035 bp and 5116 bp) which encode viral RNA and proteins which are necessary for the formation of the influenza replicon (Figure 1)
Summary
Expression of therapeutic proteins in mammalian cells is a growing field in biotechnology. Most of them were designed to be used as vehicles for gene transfer and recombinant protein expression in mammalian cells or as vaccines. These include the+sense single stranded (+ss) RNA virus members of the Togaviridae like Semliki Forest virus, equine encephalitis virus, rubella virus or Sindbis virus and members of the Flaviviridae like Kunjin virus as well as – sense single stranded (2ss) RNA virus members of the Rhabdoviridae, Paramyxoviridae and Orthomyxoviridae [3,4,5,6,7,8]. Influenza virus reverse genetics techniques developed rapidly [10] and culminated in the invention of bidirectional plasmid based systems [11] These systems are far away from being easy to handle they provide a perfect tool to evaluate the influenza A replicon for the expression of recombinant proteins. We expressed the light chain individually, evaluated the effect of using different noncoding regions (NCRs) on the expression level and investigated the influence of temperature on the influenza replicon activity
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