Transient Expression of Viral Proteins in Plants Using Agrobacterium tumefaciens
Transient expression of viral proteins in plants is a novel alternative to other expression platforms. The viral proteins can be used as potential vaccines or in diagnostics. Nicotiana benthamiana leaves or whole plants are infiltrated with recombinant Ag
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Introduction Expression of viral proteins in plants is an attractive alternative to existing expression platforms such as cell culture, yeast, E. coli, or eggs [1]. Viral proteins are often utilized as potential vaccines or in diagnostics. There are many advantages to express viral vaccines in plants such as the cost of vaccine production, no contamination with mammalian products, ability to glycosylate proteins, and the speed of production. Initially viral proteins were expressed in transgenic plants which took many months, but more recently transient expression of proteins utilizing infiltration with recombinant Agrobacterium tumefaciens has been used widely [2, 3]. One of the most important considerations before one starts this work is the choice of plant expression vectors. Various viral vectors such as magnICON® from ICON Genetics (http://www.icongenetics. com/html/02.htm) which are also delivered by agrobacterial T-DNA transfer are available. Most often RNA viruses such as Tobacco mosaic virus (TMV), Potato virus X (PVX), and Cowpea mosaic virus (CPMV) are used [4, 5]. There are vectors that use the DNA viruses such as the geminivirus bean yellow dwarf virus as a backbone, resulting in a replicating vector which also increases protein expression [6]. Another factor to consider is targeting of the protein to various plant cell compartments as that can have profound effect on protein expression levels, and a set of vectors targeting the proteins to the cytoplasm, chloroplast, ER, or apoplast are described in Maclean et al. (2007) [7]. Finally, codon usage of the gene of interest is an important factor that can have an effect on protein expression, and this needs to be determined for each gene empirically, but in general changing the codon usage
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_38, © Springer Science+Business Media New York 2016
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Inga I. Hitzeroth and Albertha R. van Zyl
to one preferred by Nicotiana benthamiana and thereby increasing the GC content to above 50 % tend to enhance protein expression. Each plant expression vector in turn needs a specific Agrobacterium strain, the choice of which depends on Ti vector resident in the Agrobacterium strain [8]. Transient expression of viral proteins in plants utilizing infiltration with recombinant Agrobacterium results in protein expression within 2–7 days. It is a fully scalable process as has been demonstrated by companies such as Medicago (http:// www.medicago.com). Plants often respond to viral infections by suppressing viral gene expression by posttranscriptional gene silencing (PTGS). Silencing suppressors are made in turn by plant viruses in response to the plant defense. NSs from Tomato spotted wilt virus (TSWV) is such a RNA silencing suppressor protein which inhibits the onset of PTGS [9]. By co-expressing a silencing suppressor with the protein of choice, protein expression can very often be enhanced. P
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