**4. The limitations and optimization of plant production systems**

#### **4.1. The problem of product shortage or the same recombinant proteins**

### *4.1.1. 3.1.1. Optimization of expression of transcripts*

**a.** Agrobacterium transformation method: Infiltration of recombinant agrobacterium suspension into tobacco leaf tissue is achieved without stable gene transfer, which facilitates the transfer of T-DNA to a very high percentage of cells, where the transgenes are expressed at a high level without a stable transfer of genes. Presently, this method has been very efficient for the production of clinical biomedicines with a fast expansion

**b.** Viral infection methods: The viral infection method depends on the capability of plant viruses, such as tobacco mosaic virus and X potato virus, which functions as a vector to convey foreign genes into plant genomes without fusing with the genome of that plant

**c.** Magnifection system: Expression systems based on viral vectors and agrobacterium methods suffer from some constraints for the co-expression of two or more polypeptides required for the production of *hetero*-oligomeric proteins [26]. Thus, a new transient expression system known as MagnICON technology has been developed by Icon Genetics Company. This method includes removing coat proteins (responsible for systemic movement) of non-competitive virus stains and systemic delivery of the derived viral vectors to all of the plants using agrobacterium as the medium of primary infection. This method not only optimizes the infection but also significantly increases proliferation, and finally results in the co-expression of several polypeptides and the rise of functional

**3. The advantages of utilizing transgenic plants as bioreactors**

Comparison of different expression systems (see Table 1) reveals the advantages of plants in

**•** The healthiness of derived products (plants cannot be the host of human pathogens and

**•** The possibility of using breeding methods and sexual crosses to obtain active recombinant multi-chain proteins (therefore, there is the possibility of producing antibodies without

**•** Reducing the costs of production (plants can produce biological materials by the use of carbon dioxide, solar energy, and inorganic materials. Moreover, the scale of production

**•** Reducing the costs of storage and transportation of recombinant proteins (when they are

**•** Removing the purification step (when the plant tissues containing recombinant protein are

**•** Capability of post-translational processing (respecting the features of eukaryotic cells).

[22-24].

28 Plants for the Future

[25].

bacterial toxins).

edible) [1].

protein production more than 100 times.

comparison with other expression systems as follows:

application of a double transformation).

can be manipulated regarding scalability).

produced in dry textures like grains).

To optimize the expression of transcripts, a widely used strategy is the use of building promoters, such as cauliflower mosaic virus 35S RNA promoter and maize 1-ubiquitin promoter, respectively, suitable for spilt-cotyledons and single-cotyledons [27]. Tissuespecific and organ-specific promoters are used for stimulating the expression of trans‐ genes (antigen vaccine HBsAgM, single-chain variable fragment Maureen G4, and Human interferon-α) in some tissues or organs, such as tubers, seeds, and fruits [28, 29]. The given specific expression of tissues prevents the accumulation of recombinant proteins in vegetative organs, which can have a negative impact on plant growth; for example, *palatine* is a gland-specific promoter; i.e., the protein is expressed in the gland but not in leaves; and also ubiquitin promoter is specified for the embryonic tissues of plants. Transcription factors (e.g., AlcR) can act as the invigorator of promoters to increase the level of trans‐ gene expression [30]. The stability of transcripts of genes can be achieved by co-expres‐ sion of the specified gene and an RNA silencing inhibitor [31].

#### *4.1.2. Optimization of translation*

Expression constructs can be designed for guaranteeing the efficiency of translation and the sustainability of transcripts. As an instance, the removal of 5' untranslated region and natural ´3 for foreign genes and introducing the leader sequence of tobacco mosaic virus RNA, RUB13 rice polyubiquitin gene, alfalfa mosaic virus, or tobacco viruses in the expressions, all, individually, have shown a significant increase in the level of transgene expressions [32, 33].

In addition to the leader sequences, expression cassettes can be designed with the AU-rich sequences in 3' untranslated regions, which may change or be removed as the editing sites for ensuring the stability of transcript. It is also proved that every organism shows codon usage deviations that may be the subject of importance for adapting the coding sequence of heter‐ ologous genes for the host gene to optimize the efficiency of translation. In this regard, the site of initial translation from heterologous protein to pair with Kozak consensus sequence, with the application of GCTTCCTCC sequence, started after codon or ACC, or ACA had been changed before that. It is better to unscientifically estimate codon changes rather than their real amount considering the changes in the expression level of transgenes in similar systems and the use of similar structures. To this end, an increase of codon combinations of (A/G)(a/c) (a/g)AUG and (A/g)(u/C)(g/C)AUG for the optimal operation of translation was, respectively, reported in Arabidopsis and rice. The given change in transgene expression could be due to the position effect, number of transgene copies, or gene silencing.

Regarding the effect of position, expression cassettes can be designed to have nuclear matrix attachment regions for ensuring the transgene insertion in proper sites for stimulating transcription factors for promoters. Furthermore, the problem of position effect can be prohibited by targeting the transgene to plastids. To optimize the production of singlecotyledon transgene, the strategies that include the use of specific genetic elements containing cAMP response elements for a simultaneous transfer with transgene in T-DNA are used. In addition, one new technology, including the structure of an artificial autonomous minichromosome, can genetically materialize excellent possibilities with several advantages, namely genetic stability due to the absence of gene silencing and position effect.

### *4.1.3. Optimization of protein stability*

To optimize the stability of recombinant proteins, known as the most important limiting factor for the function of molecular farming [34], the targeting of proteins into certain intracellular parts is demanded. The intracellular targeting not only increases protein stability but also determines the processing type of dependent protein. This can be applied for the optimization of isolation and procedures of purification by the fusion proteins and targets with high affinity [27]. Targeting of proteins can be done by the following pathways and organelles,


for the proteins that need post-translational modifications (e.g., glycosylation) [76]. The breakdown of proteins by proteases (proteolytic degradation) outside the cell is another noticeable factor for investigating the plant-based production of biomedicines.

#### **4.2. Challenge of glycosylation (protein quality)**

the application of GCTTCCTCC sequence, started after codon or ACC, or ACA had been changed before that. It is better to unscientifically estimate codon changes rather than their real amount considering the changes in the expression level of transgenes in similar systems and the use of similar structures. To this end, an increase of codon combinations of (A/G)(a/c) (a/g)AUG and (A/g)(u/C)(g/C)AUG for the optimal operation of translation was, respectively, reported in Arabidopsis and rice. The given change in transgene expression could be due to

Regarding the effect of position, expression cassettes can be designed to have nuclear matrix attachment regions for ensuring the transgene insertion in proper sites for stimulating transcription factors for promoters. Furthermore, the problem of position effect can be prohibited by targeting the transgene to plastids. To optimize the production of singlecotyledon transgene, the strategies that include the use of specific genetic elements containing cAMP response elements for a simultaneous transfer with transgene in T-DNA are used. In addition, one new technology, including the structure of an artificial autonomous minichromosome, can genetically materialize excellent possibilities with several advantages,

To optimize the stability of recombinant proteins, known as the most important limiting factor for the function of molecular farming [34], the targeting of proteins into certain intracellular parts is demanded. The intracellular targeting not only increases protein stability but also determines the processing type of dependent protein. This can be applied for the optimization of isolation and procedures of purification by the fusion proteins and targets with high affinity

**•** The intracellular parts, like *protein storage vacuoles,* have been discovered for the accumula‐

**•** Cathepsin D *inhibitor* can act as the agent of stability *of protein structures* to protect the targeted recombinant proteins in the cytosol of plants [35]. Recombinant protein production

**•** To protect proteins from cytosolic degradation, these proteins can be targeted by fusion to a C-terminal tail without a forced passing through the lumen of the endoplasmic reticulum to the membrane surface [39]. To enhance the ease of purification, proteins can be fused to *oleosin* proteins as oil bodies in order to target protein expression with the oil bodies of seeds.

**•** The proteins, like in glycosylation, that do not need post-translation modifications for their activity, can be targeted to chloroplast since post-translation modifications are not conduct‐

**•** Targeting for accumulation in endoplasmic reticulum is accomplished by two methods: one is adding SEKDEL endoplasmic reticulum signals to the end of C-protein, and the other is using fused N or C signals with y-zein. Endoplasmic reticulum is an oxidizing environment with high amounts of *chaperone proteins and low levels of proteases.* This pathway is suitable

through this signal has been proved to be very effective and economical [36-38].

namely genetic stability due to the absence of gene silencing and position effect.

[27]. Targeting of proteins can be done by the following pathways and organelles,

the position effect, number of transgene copies, or gene silencing.

*4.1.3. Optimization of protein stability*

30 Plants for the Future

tion of recombinant proteins [30].

ed in these organelles [40].

Glycosylation refers to the covalent binding of sugars to proteins in order to increase closepacking, biological activity, solubility, and biological functionality [5]. Glycosylation takes place in plants in the secretory pathway of endoplasmic reticulum and golgi apparatus. The glycosylation patterns of plants and animals differ in the composition of N-glycans; plants add residues of α (1, 3) fucose and β-(1,2) xylose to N-glycans of their protein, but animals add residues of (1 and 6) fucose, glucose, and sialic acid to N-glycans. These differences can be problematic for humans when medical animal proteins extracted from plants are used (Krupp et al., 2003); consequently, a correct human N-glycosylation demands a plant engineering. A number of strategies for changing the pattern of N-glycosylation in plants have been elaborated as following [71]:


#### **4.3. Selecting appropriate host plants**

Major economical factors in appointing an appropriate host include the total biomass yield, storage characteristics, ease of transport, value of recombinant proteins, maintenance costs, its availability for workers, required area, duration of production cycle, cost of subsequent products, and edibility [27, 34]. In addition to the economical analysis, a sufficient host should be appropriate in terms of transformation and regeneration [34, 72]. In addition to the high potential of tobacco for transformation and regeneration, it has the majority of the aforemen‐ tioned economic benefits [27, 41, 42]. However, tobacco (except the *cultivar 81 V9*) [43] contains high amounts of toxic combinations, nicotine and other alkaloids, that cannot be removed during the purification process. In spite of this, alternative forage crops like alfalfa and lettuce are being investigated and discovered as a suitable host for molecular farming [44]. However, forage plants generally suffer from the problem of instability of expressed proteins, by which drying and freezing of the leaves and immediate processing following the harvest have been inevitable [27]. The seed-based expression of proteins is considered to be more ideal regarding the fact that it neither affects the growth of plants nor needs the freezing of leaves or immediate processing after harvest, and it allows the long-term storage of proteins at a limited tempera‐ ture without decreasing the level of activity [45, 46]. In this regard, grains, especially rice and corn, have been cited as the superior ones. Maize has abundant advantages, such as having the highest rate of biomass yield among food crops and ease of transformation and production increase [47]. The high amount of protein (20%-40%) in the grains of legumes with remarkable levels of self-pollination in soy and peas is the main reason for transgenes of these plants for protein accumulation [48-50].
