Optimization of transgene expression in host plant

Plant as expression systems offer many benefits over conventional systems. However, there are still many challenges that remain to be overcome to use plants as main stream production platform. For the development of plant-based production platform, one needs to optimize the expression level of a recombinant protein.

Optimization of promoters and terminators

In order to optimize transcript expression, the general strategy is to use strong and constitutive promoters, such as the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and maize ubiquitin-1 promoter (ubi-1), for dicots and monocot, respectively (Fischer et al., 2004). Organ and tissue-specific promoters are also being used to drive expression of the transgenes in the tissue or organ like the tuber, the seed and the fruit (He et al., 2008). This tissue specific expression therefore prevents accumulation of the recombinant protein in the vegetative organs, which might negatively affect plant growth and development. Additionally, inducible promoters, whose activities are regulated by either chemical or external stimulus, may equally be used to prevent the lethality problem (Corrado and Karali, 2009), as it is being used in cell suspension cultures (Nara et al., 2000; Peebles et al.,2007). Besides, transcription factors can be used as boosters for the promoters to further enhance the expression level of the transgenes (Yang et al.,2001). Moreover, it has been recently found that the terminator of the heat-shock gene of Arabidopsisthaliana shows an increase in transcription of a foreign gene by fourtimes (Nagaya et al., 2010).

Furthermore, the expression constructs can also be designed to ensure transcript stability and translational efficiency. This involves, for instance, the removal of the native 5′ and 3′ untranslated regions from the foreign gene and introducing 5′ untranslated leader sequence of the tobacco mosaic virus RNA, rice polyubiquitin gene RUB13, alfalfa mosaic virus or tobacco etch virus in the expression construct, all of which have been separately shown to significantly enhance the expression levels of transgenes (Lu et al., 2008; Sharma et al., 2008). 5′ UTR of rice poly ubiquitin gene RUBI3 along with its promoter was reported to enhance the expression of GUS at mRNA level as well as translational level suggesting that 5′ UTR plays an important role in gene expression (Lu etal., 2008; Samadder et al., 2008). The untranslated leader sequences ofalfalfa mosaic virus mRNA 4 or tobacco etch virus have been found to enhance the transgene expression by several folds due to enhanced translational efficiency of transcripts (Datlaet al., 1993; Gallie et al., 1995). In addition to these leader sequences, the expression cassette design such that the false AU-rich sequences in the 3′untranslated regions that may act as splice sites are removed or modified, to ensure transcript stability (Mishra et al., 2006). Besides, the transcript stability can be ensured by co-expressing the gene of interest and a suppressor of RNA silencing (Voinnet et al., 2003). It is also established that each organism exhibits biased codon usage, such that it might be important to adapt the coding sequence of the heterologous gene to that of the host plant in order to optimize translation efficiency (Lienard et al., 2007). In this regard, the translational start-site of the heterologous protein is modified to match with the Kozak consensus for plants (Kawaguchi and Bailey-Serres, 2002) or by using the sequence GCT TCC TCC after initiation codon, or ACC or ACA before it (Sharma and Sharma, 2009).

Optimization of codon usage

The codon modification, however, should be empirically determined rather than predicted because of the variation in the levels of transgene expression in the same system, using the same construct (Rybicki, 2009). To this end, codon combinations (A/G)(a/c)(a/g) AUG and (A/G)(u/C)(g/C) AUG have been reported to be optimum for enhanced translational activity in Arabidopsis and rice, respectively (Sugio et al., 2010). This variation in the expression of the transgene may be due to position effect, copy number of the transgene or gene silencing. With respect to position effect, expression cassettes can now be designed to the nuclear matrix attachment regions (MAR), which are regulatory sequences that ensure the placement of the transgene in suitable regions for mobilizing transcription factors to the promoters (Streatfield, 2007). Besides, the problem of position effect can be avoided by targeting the transgene into the plastids (Cardi et al., 2010). For optimizing the generation of single-copy transgenics, the strategies that have been used include the use of specific genetic elements, including the cAMP response elements (CREs), for co transfer with transgene in the T-DNA (De Paepe et al., 2009). Additionally, a new technology, which consists of the construction of genetically autonomous artificial mini chromosomes, has been described as providing infinite possibilities with several enormous advantages including gene stability; owing to the absence of gene silencing and position effect (Ananiev et al., 2009).