Journal of Plant Bioinformatics and Biotechnology 2021, Volume 1, Number 2

Review Article

Using Molecular Farming Technology in Producing

Human Proteins in Plants
Farzaneh Fazeli
Department of Biology, Payame Noor University, P.O. Box 19395-4697, Tehran, Iran

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article online Citation Fazeli F. (2021). Using Molecular Farming Technology in Producing Human Proteins in
Plants. J. Plant Bioinform. Biotechnol., 1(2): 114-128.

[Link]

ABSTRACT

Article info Early progressive in plant genetic and recombinant DNA technology, has helped
Received: 2021-10-24 to improvement and support of researches in identity and manipulate of genetic
Accepted: 2021-11-04 of enzymes in path of produce of seconds metabolites. The mean of Molecular
Available Online: 2021-12-13 farming is produce of drug biology and important proteins with plants. Plants and
ID: JPBB-2110-1016 molecular farming have high potential in infinite produce of recombinant
Checked for Plagiarism: Yes proteins (vaccines, antibodies etc). Since 1986 that produced of first recombinant
Peer Reviewers Approved by: drug protein in tobacco, has produced the large number of proteins medical need
Dr. Bahman Fazeli-Nasab in plant and in the present has acceptable produced of recombinant proteins in
Editor who Approved Publication: world. Proteins has produced in plants are devoid of harmful end tocsins, human
Dr. Behzad Ghareyazi pathogeneses, animal malady ingredient and virus and bacteria DNAs that there
were in current expression systems such as animal and prokaryote cells culture
and as the result of are ideal model for proteins of expression and identity. Plants
Keywords: are an economic system for a large of protein produced with present of transgenic
Molecular Farming, Transgenic plants stable. It is useful and economic for large produced and increase plant
Plants, Biotechnology, genetic potency as identity of meticulous and quickly of genotypes that produce
Recombinant Protein. extreme of production using biotechnological methods

animal cells and animals, the process of

M
1. Introduction
producing and using such compounds was
olecular farming which is a new somehow limited; however, significant
industry in agricultural biology advances have occurred in the 1990 s on
refers to producing plants used for molecular biology and plant biotechnology. The
producing external proteins and production, processing and use of recombinant
pharmacological and aromatic compounds in proteins produced in plant tissues using
the genetic plants. Since ancient times, man has molecular farming techniques have increased
always used plants as drugs, yet recent significantly. By combining genetic engineering
applications of transgenic plants and their and traditional plant breeding, it is possible to
derivatives for producing molecular drugs such transfer beneficial genes to plants. In turn, it
as antibodies, enzymes, growth factor, and can while transferring genes such as resistance
plasma proteins have had a remarkable to pests, diseases, and environmental stress can
development. Due to problems available in increase crop yields (such as herbal medicines)
producing such compounds and proteins in or induce transgenic plants to produce human

*Corresponding Author: Farzaneh Fazeli (Farzanehfazeli2020@[Link])

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drugs and vaccines [1]. frequently produced from the organs of the
whole plants [2].
Using medicinal compounds with a plant
origin has a long history. Moreover, due to the Over time, the ways of using plants for
side effects of chemical drugs and producing recombinant proteins with high
insufficiencies of modern medicine in treating values ranging from herbal treatments to anti-
some diseases over time, a sudden explosion drug products such as antibodies, vaccine anti-
has occurred in growing and producing herbal genes, enzymes, growth factors, research or
plants and manufacturing various drugs and diagnostic reactions, and cosmetics. Recently, it
vaccines by plants. According to predictions has expanded so incredibly that has caused
made by the world health organization (WHO), significant revolutions of paradigms in the field
over %80 ofthe world’s population (about 5 of pharmacology [3]. Along with rapid
billion people) still prefer to cure their diseases development of technology in recent decades,
by herbal remedies. Nearly a quarter of the the molecular farming industry has significantly
world’s manufactured drugs have plant origins developed to help people in various fields. The
either extracted directly from plants or advantages of using a plant platform in the
modified and synthesized based on an herbal industry of molecular farming rather than other
composition. Research on traditional medicine platforms are given in Table 1 [4-8]. Some key
and using herbal plants is carried out across the points of popularity of plant platform systems
world, especially in India, Japan, Pakistan, Sri are ease of cultivation, low cost, little/ no
Lanka, and Thailand. pathogenic pollution, high speed of mass
production of recombinant proteins, plant’s
Among the European countries, Albania,
ability to assemble and produce protein
Croatia, France, Germany, Hungry, the
complexes along with post-translation
Netherlands, Spain, England, and Turkey use
variations similar to eukaryotes (PTMs).protein
1,500 species of medicinal and aromatic plants.
twisting is the most fundamental feature of
Around 1,400 herbal products are produced in
proteins for survival and activity of
Europe and the U.S. In fact, a number of popular
recombinant therapeutic proteins. However,
medicinal products are taken from plants. For
because of the complexity of protein processing
example, the most popular painkiller, aspirin, is
process and capacity limitations of PTM
derived from the plants, Salix and Spiraea.
systems, properly twisted proteins are not
Moreover, anticancer drugs such as
achievable in the prokaryotic expression
Paclitaxeland Vinblastine are directly produced
systems [9].
from herbal resources. In addition to the
growing consumption and prescription of In general, plants have the ability and
medicinal plant products, approximately 79% capacity of assembling PTM platforms to make
of today’s prescribed drugs include at least a large molecular proteins with high biological
chemical which has plant origins. Demand for activities; however, they are unable to process
expression and manufacture of pharmaceutical the mechanism of the glycosylation.
proteins has also increased considerably. It has Fortunately, this shortage is compensated with
in turn caused expansion of manufacturing the contribution of bioengineering
industries of these proteins and other related achievements which aim to synthesize both
systems. Moreover, it is indicated that in some human and non- human structures, and in turn,
cellular lines, the secondary metabolites such as increase manufacturing of homogeneous
Alkaloids, flavonoids, and terpenoids are more products with higher quality and greater
quantity [10].

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Table 1. Available platforms for expression of recombinant proteins with strengths and
weaknesses of each platform [11]
Expression
Advantages Disadvantages
System
Possibility of easy manipulation
Low cost
Expression in large quantities Improper folding
Easy to scale Lack of post-translational
Bacteria short time manipulation
Ability to adjust production steps and Accumulation of endotoxins
amount High production value
Suitable twist and has the possibility of
changes after translation
Expensive facilities and cell
culture medium
adjustable features
Mammalian Difficult to destroy cells due
Rapid and incremental growth
cells to having a hard and thick
Easy to reproduce
wall
Time consuming and costly
Easy and low cost of its culture medium
Protein hyperglycosylation is
Its recombinant proteins are capable of
Yeast limited by glycosylation
post-translational modification
capacity
High level of expression
Ability to produce complex proteins such
as secretory proteins; Membranes and
Expensive culture medium
intracellular proteins
Insects’ cells and requirements of its
Suitable for twisting and post-
culture medium
translational changes
Fast and doable
Optimal growth conditions
Free of pathogenic and harmful bacterial
Process complexity
agents
plants Limited glycosylation
Economical and economical
capacity
Ability to post-translational modifications
such as mammalian systems
2. Genetic engineering cause diseases such as Crown Gall Tumour and
Hairy Root. Studies showed that the hairy roots
Genetic engineering is a multi-facet science, and
made by a species of this bacterium, A.
recent developments in plant genetics have
Rhizogenes have a suitable tissue for producing
made great contribution with improving and
secondary metabolites. As a result of the high
enhancing research in production of secondary
resistance and productivity of these tissues in
metabolites. Much of literature on secondary
hormone- free culture conditions, a large
metabolites has concentrated on genetic
number of medicinal species have so far
identification and manipulation of enzymes
changed by using this bacterium among which
involved in the metabolic pathway of a
are cultivation of hairy roots of the herbal plant
secondary metabolite synthesis. A natural tool
Artenisia Annua for manufacturing an active
used in the process of genetic engineering
drug compound [12].
found in most plant species and especially in
dicotyledonous plants is a soil bacterium Transgenic plants can act as biotic
known as Agro bacterium. Various species of bioreactors used for cheap manufacturing of
this bacterium include natural engineers which

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chemical and medicinalproducts. This process agriculture, have offered the possibility of
is called molecular farming. The fourth- producing a number of vital molecules (such as
generation transgenic plants are those which drugs, insulin, and blood proteins) and
are produced for medical and hygienic important hormones (such as growth hormone,
purposes. Increasing demand for biologic drugs vaccines, and interferons). Today, there is a
along with their significantly high price has globalconsensus for production of these
limited their access, so precise planning is recombinant proteins through plants [1, 14].
required to supply large quantities of them with
more reasonable prices. In fact, plant genetic Innovations in plant molecular engineering
engineering which seeks to produce biologic technologies have also provided a variety of
drugs is a new field which has a lot of potential plant-based production platforms, ranging from
benefits. cell culture to growing plant tissues under
sterile conditions and growing the entire plant
3. Production of recombinant proteins in in a glass or field [15].
plants
The biological production platforms are
Recent advances in agricultural classified into three groups: (1) laboratory
biotechnology covers three major waves: (1) culture systems including cell suspensions,
the wave of gene transfer in plants aiming to hairy roots, and moss protons, (2) aquatic
resist pests and diseases (2) the wave trying to plants (such as duckweed and microalgae), and
increase crop productivity (3) the wave of (3) whole plants using both stable and transient
molecular farming. The third wave regards methods. The stable expression method of
cultivation of plants as a biological plant which whole plants also covers both leaf-based and
is used to manufacture various proteins, seed-based systems. The characteristics of each
especially human proteins for medicinal, platform and their strengths and weaknesses
scientific and industrial purposes. are described below. A comparison of cost,
The biological systems which make up application, production time, scalability, and
recombinant proteins include different kinds of compatibility adjusters for different operating
bacteria, yeasts, animal cells, insects, plants, systems is shown in Table 2.
and cells from genetically engineered animals.
An expression system should be able to possess 4. Methods of protein production through
high production characteristics, maximum molecular farming systems in whole plants
biological activity, ease of purification, and
The expression method in whole plants still
minimum production time for the recombinant
dominates plant-based bioproduction platforms
protein. Due to the following characteristics,
and transient expression methods through viral
plant expression systems are superior to other
and non-viral vectors or fixed transfer methods
expression systems: plant systems are much
which rely on trans-targeting nucleotrans or
more economical than industrial systems
chloroplasts. It is considered as a suitable
(bioreactors or fermenters), the number of
transient platform for the production of vaccine
recombinant products produced in plants can
antigens or monoclonal antibodies against
be equal to and even greater on an industrial
some diseases (Figure 1). However, transient
scale, lower risk of contamination with
and fixed expression methods in the whole
pathogens. Moreover, plants can be tilted so
plant which lead to establishment and
that the synthesized proteins are directed to the
maintenance of stable transgenic lines are
intracellular organs with more stability. Even,
extravagant and time consuming. Moreover,
these proteins can be expressed directly in
some other problems are gene silencing,
certain organs (such as chloroplasts) [13].
situational effects, and GMO environmental
Thus, recombinant protein production concerns [17]. Today, there are 4 methods of
methods which act as the most fundamental protein production which can be used in
methods in biotechnology and molecular molecular farming systems:

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Table 2. Comparison of bio-production platforms based on different plant bases [16]

Time Type of
Process
Type of required protein Productio
Plant type Plant species complex
platform to can be n valueb
ity
producea harvested
Cell Tobacco, rice, Therapeuti
Laboratory 7-14 days average Easy
suspension carrots c proteins
culture
Physcomitrella Therapeuti
system Moss 14-30 days Average Easy
patens c proteins
Therapeuti
Lemna sp., c proteins,
Moss 20-40 days low Average
Aquatic Spirodela sp. Industrial
plants enzymes
(hybrid Chlamydomona
system) Phytoplank s reinhardtii, Therapeuti
14-30 days low easy
ton Dunaliella c proteins
salina
Whole plant Stable
3-6
(leaf-based transgenic Tobacco, rice biopolymer Very low Difficult
months
systems plants
and seed-
Transition N. benthamiana, Therapeuti
based 2-7 days Low Easy
plants lettuce c proteins
systems)
a: Time required to collect the maximum amount of recombinant protein in a culture system after incubation
b: compared with mammalian cell culture platform

Figure 1. Schematic representation depicting the application of transient expression system for the
production of some recombinant proteins [18].
4.1 Sustainable expression method Sustainable nuclear transfer: To date, this
method has been the most popular method
Sustainable expression method which is
among crops which has built almost all
done in two ways: stable nuclear transfer and
products available at the market. In this system,
stable plastid transfer. The latter is used in
a method of transferring the foreign gene into
most agricultural crops.
the plant cell is required. This method is usually
incorporated with Agrobacterium tumefaciens
or particle bombardment into the host nucleus

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genome in a stable manner [19]. Advantages: in 4.3 Sustainable transfer of species grown in
some crops such as cereals, the produced hydroponic medium
protein first stored in the grain is harvested in
In this system, the transgenic plants including
the dry state, and then, it is stored until
encoding gene of the external protein which are
processing. For example, the moisture content
grown in hydroponic medium release the
in driedcorn kernels is % 11-15, yet, at this
desired product as a part of the root secretion
level inside the grain, it remains neutral;
in this culture. Advantages: the plants grown in
therefore, if non-refrigerated storage is done
the hydroponic medium are placed in the
properly in long term, the protein can remain
greenhouse; therefore, the risk of
undamaged for two years. Another advantage is
environmental contamination reduces, and in
the large volume of crop with the least cost.
turn, the purity of the desired product is
Disadvantages: Some cereals, such as corn, have
significantly greater. Disadvantages:
the potential to intersect with native species or
manufacturing great amounts of protein is
other crops.
probably impossible, and the facilities of the
Stable plastid transfer of crop species: This hydroponiqueculture are really expensive [22].
method was first reported by Wab et al. (1990)
using tobacco, tobacco is the only species on 4.4 Gene expression in cell culture medium
which plastid transfer has been routinely (suspension cultures)
performed, the protein expression level in this A branch of biotechnology is tissue culture, and
method during chloroplast transfer has been its application in producing and increasing
reported to have 40% of dry weight [20]. Since drugs in plants is worth to evaluate.
plastid is not usually transmitted through Historically, the technique “tissue culture “was
pollen grains, external crosses are not a major first used from 1939 to 1940 in plants, yet it
concern. Disadvantages: As protein stability was in 1956 that a US pharmaceutical company
changes in any tissue over time, even in the in the U.S. (Pfizer Inc) introduced the first
refrigerator, extraction and purification must invention used in producing metabolites using
be performed at very specific post-harvest mass culture. So far, a great amount of visnajin
times. Tobacco is currently a controlled product and diosgenin compounds have been produced
and is not edible, so large-volume products and using tissue culture rather than the natural
oral vaccines are not feasible using this system state (extraction from the whole plant). In fact,
[15]. plants are a rich source of chemicals which are
4.2 Method of transfer expression of crop used as medicinal compounds. Products
species obtained from the secondary metabolism of the
plants are among the most valuable plant
This method depends on the ability of some
chemical compounds. Using the tissue culture,
plant viruses such as the tobacco mosaic virus
the secondary metabolites can also be produced
(TMV) and the potato virus (PVX), which is
in vitro. Noteworthy, secondary metabolites are
used as vectors to transfer foreign genes into
substances including complex acids, lactones,
the plant with no fusion. The centrifuge is
flavonoids, and anthocyanins that are vastly
collected under vacuum Advantages: The TMV
used as plant extracts or powders in treatment
virus can be easily manipulated to speed up the
of many common diseases [23].
contamination process, small amounts of this
target protein can be obtained in a few weeks The most important strategies which can
Disadvantages: it is not probably suitable for increase the production of plant secondary
the required protein in high quantities. Also, in metabolites through tissue culture use biotic
this method, the product should be processed and abiotic stimulants that can affect the
immediately for storage so that the plant tissue metabolic pathways of secondary metabolites
is not destroyed. Tobacco is a controlled synthesis, increase their production rate,
product and does not grow in large areas [21]. promote the initial compound suitable for the
culture, build new genotypes through

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protoplast fusion or genetic engineering, use valued in biological pharmacology industry

mutagenic materials to produce high-yielding [25].
varieties, cultivate the root tissue of medicinal
plants (since the root has a greater potential for Due to ease of protein recycling and low
production of secondary metabolites than other costs, the cultivation of hairy roots is
plant tissues) [23]. considered as an alternativesystem for
producing recombinant proteins. The
5. Using bioreactors in industrial production recombinant proteins of transgenic plant’s
of secondary metabolites roots secret rhizoid secretions in VIZ culture, so
there is the possibility of continuous production
Production of plant secondary metabolites and recycle of protein from the culture,
which has numerous medicinal chrematistics in regardless of lysis and cell destruction while
laboratory conditions has more advantages extraction. Moreover, recombinant proteins
than extraction of these compounds from plants produced through root cultivation culture
under natural conditions. Precise monitoring of method can improve the quality and quantity of
different parametersprevents any change in proteins without processing, which in turn,
material quality over time whereas in natural reduces the production costs [26]. In a recent
conditions, it is constantly affected by weather research, applications of the hairy root
changes and pests. A number of studies have cultivation method for producing protein have
been carried out on usingsuspension cultures been deeply discussed by Gutierrez-Valdes,
and plant cell in manufacturing secondary 2020 [27].
metabolites. Among instruments used for vast
cultivation of plant cells are bioreactors. Infact, 6. Types of recombinant
bioreactors are the most important instruments proteinssynthesized in plants
used in commercial production of the
secondary metabolites through Although proteins are commonly used in
biotechnological techniques [24]. research, medicinal, and industrial fields,
extracting proteins from natural resources is
Usingbioreactors in mass production of difficult and costly, so plants are used for
plant cells has numerous benefits, mainly producing large quantities of transgenic
because of presence of better and more precise vaccines and proteins. Meanwhile, blood
control over special conditions required for products, hormones, enzymes, antibodies, and
industrial production of bioreactor compounds vaccines are successfully produced in plants.
through cell suspension culture, possibility of Today, considering molecular farming
stability during different stages of cell culture in purposes, the proteins built in plants can be
the bioreactor, easy transfer and movement of divided into 5 groups:
cultivation, high volume of cell reproduction,,
and consequently, significant increase of the 6.1 Main drugs and pharmaceutical
final product (bioactive compound). Due to the intermediates
fact that bioreactors provide the optimal Main drugs including all proteins used
condition for production of the secondary directly as the drug along with proteins used in
metabolites from plant cells, a lot of changes manufacturing pharmacological compounds
have occurred to optimize these systems for such as thrombin and collagen (treatment) and
producing compounds with medicinal value trypsin and aprotinin (mediator).
(with plant origin). The first poultry vaccine
approved by USDA and the first recombinant 6.2. Antibodies
herbal medicinal protein, Elelyso, licensed by
FDA were produced respectively in suspension These are glycoproteins, which due to their
of the tobacco and carrot. This clarifies the high specificity, can recognize the target gene
significance and competitiveness of plant and stick to it. This specific and individual
suspensions in manufacturing proteins highly activity of attachment provides the opportunity
of using antibodies for various usages such as

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identification, prevention, and treatment of antibody parts of fragment variable (FV). These
some diseases. To provide such antibodies in antibodies can be produced both in glycine/
mammals, two different types of cells are non- glycine forms in plants [28]. For example,
required, yet as far as plants can express four mass IgG purification from tobacco plant is
different transferred genes; it is possible to relatively easy. Some of recombinant antibodies
accumulate these antibodies in a single cell. made in plants given in Table 3.

This group includes all antibodies (the

secretory antibody of IgA, IgG, IgA) and
Table 3. recombinant antibodies made in plants
Plant mAb form Recombinant antibody
A. Rabidobsis IgG (Kg) Fungal cutinase
Soy scFv Anti-herpes simple virus 2
Potato scFv Ubiquitous
Tobacco scFv Abscisic acid
Tobacco scFv TMV
Tobacco IgA Streptococcus mutant
Corn, potato, tomato, and
S protein SARS-Cov-S
tobacco
6.3 Vaccine antigens 6.4 Enzymes
The main ingredient of each new serum This group includes hydrolases, glycosylases,
includes dead microorganisms, non-pathogenic and proteases. The Oxidation-reduction
microorganisms, and microbial products such enzymes such as lactase, the enzymes used in
as toxin or purified microbial parts. The vaccine white fiber and bioglue of wood products all
of the antigenic materials provides immunity represent a distinct class of industrial enzymes.
against a disease. In fact, antigens are materials The enzymes involved in biomass transform are
which equip the immunity system to produce other alternatives used for ethanol production
specific antibodies. Such stimulation makes the in molecular farming.
immunity system to destroy bacteria
andviruses entering the body. Although 6.5 The most significant recombinant
vaccines are mostly harmless, some may rarely proteins
cause pathogenic infections. The first medicinal protein synthesized in
Development of vaccine subunits based on transgenic tobacco, in 1986, was human growth
the use of a specific protein subunit of a hormone. Since then, many human proteins
bacterial or viral pathogen has shown a lower have been expressed in plants. Some proteins
risk of side effects than complete bacterial or built in plants are Insulin, Insulin-like growth
viral vaccines. Vaccines whose active ingredient factor Erythropoietin, interferon, aprotinin,
is recombinant antigens lack this small risk. In Leu-enkephalin and somatotropin.
the past, the recombinant protein subunits of
6.6 Insulin
vaccines were usually produced in Coli.E, yeast,
or mammalian cell culture, but these The molecule of insulin was the first
manufacturing methods have been limited in medicine upon which was created its changes
recent years by scale, product quality, and cost. and improvement. In the past, insulin was the
Tobacco which acts as an alternative expression result of extraction and purification of the
system is used for building the subunit pancreas. This kind of process had at least three
production of the recombinant vaccine. So far, advantages. First, there is a serious limitation
many vaccine proteins have been produced in for their extraction. Second, this type of
tobacco, some of which are the vaccines extraction is expensive and time- consuming.
synthesized for cholera, hepatitis B virus, acute Third, in cases, there is a reaction to intolerance
respiratory syndrome (SARS), corona, and HIV. in this kind of allergy products. These

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limitations led to numerous researches carried mellitus, and is also used for treating patients
out on basis of isolation of the gene producing with defects in insulin receptors [30]. Insulin-
human insulin. Then, the desirable gene was like growth factors (IGFs) include IGF-1, IGF-2,
inserted in E. Coli or genetic engineering, and and two receptors, (IGF-1R, IGF-R), in cell
finally resulted in producing human insulin membrane. Moreover, there are six attachment
through recombinant DNA technology. In fact, proteins called IGFBP1-6. Thefactors, IGF-1and
this technology provided human insulin which IGF-2, are in fact polypeptides with the
is cheaper, more available, and less risky and molecular weights of 7649 and 7471.
has greater industrial immunity. Structurally, both types of IGF are similar to
insulin in terms of having two chains (A, B)
Recently, one of the most remarkable connected by di-sulfide bands. IGF-1 and IGF-2
achievements of genetic engineering and are homologue by 62 %. Also, they have 50%
biotechnology has been the manufacturing of homology with insulin, and have their
medicinal recombinant proteins among which metabolic effects resemble those of insulin. This
the most commonly- used one is insulin. Due to homology reflects a similar range of biological
some side effects of Exubera, such as lowered activities. IGF-1 protein can also affect growth
performance of inspiratory system, a sudden regulation, survival, and physical metabolism.
decrease is reported in its demand. As a result,
manufacturing of inhaled insulin has dropped 6.8 Recombinant human insulin- like growth
significantly. Patients with diabetes type II are factorsrhIGF-1
the major consumers of edible drugs of anti-
diabetes. Since a great number of people across Recombinant human Insulin Like Growth
the world suffer diabetes typesI and II, they Factor-1 was first produced by recombinant
need more available ways to access insulin DNA method in 1986. Rice is one of the best
derivatives. To solve these problems, several plant species which can be used for producing
methods such as taking insulin capsules with this protein, since it is free from any harmful
nanoparticles and using proteas inhibitors were chemical ingredients such as toxic alkaloids like
invented to prepare insulin derivatives. nicotine in tobacco. Also, it has low sensitivity.
Transferred to rice through gene transfer by
Oral vaccine is one of the best immunogens Agrobacterium. Itwas proved by in vivo studies
which stimulates the mocusous response of the that human recombinant IGF-1present in
immunity system. The proinsulin extracted transgenic rice could reduce blood glucose level
from the culture of plant tissues which are also in fat rats with diabetes II and rats with
among the best compounds for injection of diabetes I. these studies confirmed the biologic
insulin and its derivatives can be used both on activity of human recombinant IGF-1produced
animal and human samples. To meet this high in rice [31].
need, production of human proinsulin in the
plant of strawberry can be considered as a IGF 1 Expression levels in transgenic rice
beginning point for cheap and constant and tobacco are 22- 132 ng/mg, that is, about
production of insulin [29]. Moreover, potato 0.002-0.011 % of all solvable proteins. In this
can be used for expression of insulin gene. The study, it was proved thathuman insulin- like
content of insulin taken from potato cultivation growth factors -1 expressed in the chloroplast
is reported to be 0.05% of TSP. the method of of the transgenic tobacco resembles natural
tissue culture is a successful and efficient way protein, and is quite practical [30].
for producing proteins such as insulin [29].
6.9 Aprotinin
6.7 Insulin-like growth factor Aprotinin is a Serine group protease
The insulin-like growth factor plays a key inhibitor protein (including trypsin,
role in reproduction, cell discrimination, chymotrypsin, kallikrein, and pepsin). This
growth and apoptosis. This protein acts as a feature of Aprotinin modulates and reduces the
treating factor forInsulin-dependent diabetes systemic inflammatory response (SIR) in some

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surgeries acts as a Cardiac Respiratory Bypass cells. Sometimes, buildingerythroprotein in the

Surgery. Besides, it reduces bleeding. In the body reduces in some pathological conditions
past, the initial ingredients for producing such as Kidney parenchyma damagewhich leads
commercial protein A were extracted from 6 to different levels of anemia. This hormone is
cows. As a result of significant increase of composed of a protein and a carbohydrate part.
sponge- like Encephalopathy(BSE) and other
animal pathogens in cows, high expense of EPO recombinant proteins are mostly used
surgery and anesthesia of cows as well as in treating different kinds of anemia. This
limitations on producing natural protein A from protein is one of the first recombinantcytokines
cows, the new hypothesis of preparing cheaper produced in plant cellsdue to heavy weight of
and more accessible protein A was proposed for EPO molecule (30 kDa), free migration of this
its manufacturers. Due to the great significance protein through the cell wall into the culture is
of BSE, the protein A manufacturers were difficult, so EPO secreted into the protoplasts of
encouraged to build this product from dry the tobacco plant [Link] EPO
materials; thus, it was called BSE- free. The synthesized in plant cells are glycosylated in
main advantage of using such plant resources shape, and resemble its natural form, but the
was removing the fear of mammal pathogens location and length of its polysaccharide differ.
from the initial materials. Unlike initial Unfortunately, this recombinant product is
materials taken from 6 cows, these plant quite rare (0.0026% of the total soluble
resources were free from viruses and prions. protein). The gene expression of EPO protein
Moreover, because of using plant seeds for occurs with very high efficiency in protoplast
producing Aprotinin, other limitations of dry (moss Physcomitrella patens (Hedw).
matters were also removed. Moreover, it is done quite well in the whole
plants of Arabidopsis and tobacco [26].
The first attempt to produce Aprotinin from
the transgeneic seeds of corn which was 6.11 Interferon
reported by Zhong et al. (1999) [32] indicated Interferons are glycoproteins which are able
the expression of this protein as %0.068 of total to stimulate the immunity system against
protein solvable (TPS) from a line with 20 various pathogens and tumors. In fact,
copies of [Link] study by interferons are cytokines which are responsible
Delaney (2002) [33], the protein harvest was to antivirus responses. Recombinant
reported with efficiency of over %8.9. Later, interferons with plant origins are sometimes
Zhong et al, (1999) [32] tried purification of used for treating Kaposi’s sarcoma, myeloid
Aprotinin in cases such as Aprotininextracted leukemia, and hepatitis types A and C. gamma
from tobacco which wascommercialized by interferon belongs to the interferons class II
Sigma-Aldrich Company (St. Louis, MO, USA) to which is in charge of regulating the response of
reach an efficiency of over %50 [34]. the immunity system to tumors and viruses.
Interferons are frequently produced in plant-
6.10 Erythroproteins
based systems. The first report of producing
Hematopotins are among the first human interferon dates back to the early 1990s
recombinant cytokine drugs. Human [35].
erythroprotein (EPO) is a glycoprotein
hormone which is responsible for regulating Human interferon is mostly produced in
the content of red cells in mammals’ blood. transplastomic of the tobacco plant. It is
Erythroprotein is mostly produced kidneys, but estimated that its productionefficiency is %20
it is rare liver. Its production and secretion of the total solvable protein. Besides human
isstimulated and increased by reduction in red interferon, the pig, hen, and salmon types are
cell levels as well as reduction in oxygen successfully produced in plant- based systems.
delivery to the [Link] acts on Gama interferon is used in rice cell cultivation
Erythrocyte precursors, and is in fact, the suspension using corn promotor. Alpha-
discriminating factor in Erythrocyte mature amylase peptide is used as a signal to secrete

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interferon-gamma produced in culture Zmapp to cure Ebola in 2014 in west Africa (9 g

[Link]-2b (Locteron) recombinant per patient) [41].
protein which is built in the
plantduckweedLemna minor L. is used in great 8. Other proteins
volumes to produce monoclonal and interferon For the purpose of providing positive and
antibodies [36]. negative immunity, it is possible to use
antivirus proteins in the infectious phase of a
7. Corona disease and molecular faming
disease. In this way, other side effects of the
The corona virus II (SARS- CoV-2), a new Covid 19 such as Cytokinin cascade which are
virus which causes acute respiratory syndrome, built in the first phase of the infection are
created a global pandemic in 2020, and in turn, controlled. Among the inhibitors of lectin virus
resulted in an increasing demand for solving is specifically an important protein synthesized
the problem and carrying out research on this by the molecular farming method. Some
issue by the WHO and the research centers [37]. reasons of significance of lectin are the
Under such adverse conditions a double possibility of its natural synthesis in most
pressure was imposed on both hospitals and excellent plants and its reversible attachment to
health centers. Accordingly, researchers and carbohydrates.
research centers carried out great studies and
experiments to produce drugs and vaccines Due to presence of glycan in the surface of
suitable for this disease. The Corona virus also the viruses, lectin canact as an antivirus to stop
clarified the necessity of implementing long the infectious cycle of some viruses such as
term organization and research on a proper, AIDS virus, Cytomegaly virus,
fast, flexible, and multi- facet platform for all respiratorysystem viruses, influenzas type I
medicinal requirements (including diagnosis, virus, and the corona virus. Besides, so far
vaccine, medication) for emergency cases of twenty species of lectins have been identified in
bioterrorism-related infectious diseases [38]. different plants which can stop the infection
caused by the corona virus. This is possibly
Plants can be used as a suitable system for done through random attachment to glycan in
production of Corona anti-virus antibody S- protein virus. One of the best options for
protein. Inplants, transfer expression method is producing plant lectin is thealgae Griffithsin, yet
mostly used along with virus vectors to make the toxic level of its stenolid is limited for
anti- covid 19 antibodies. The average rate human cells, yet in tobacco pant, lectin is
reported for producing antibody in the transfer produced by an average of 760 μg/ [Link],
method of molecular farming is 870 μg, which other biologic medicines which are used for
can be a very desirable output for systems and Covid 19 are Anticoagulation therapy [42] and
factories which need these products. These Imonodolitors which stop the pathways of the
products and efficiency of the molecular Anticoagulation therapy, cytokinin [43].
farming methods are really useful and practical Molecular farming can specifically reduce the
especially in situations like the current time and cost of producing the Anticoagulation
outbreak where a great quantity of antibodyis therapy drugs, Thrombin and fibrinolytic. Also,
immediately required. the antibodies, immunomodulators, can be
successfully being produced in plants.
In this pandemic, Italy was the first country
which had the highest rate of the corona- 8.1 Corona virus
infected patients [39]. In the mid Mars, 2020,
around 9-11 % of patients suffering Covid 19 As the Sars-cov-2 genome sequence was
were hospitalized in ICUs [40]. In an identified and published, hundreds of industrial
experiment [40], it was estimated that the research universities and groups seeking to
rateof antibody required for this demand in inventthe corona vaccinelaunched theirstudies
Italy for all ICU patients was relatively similar relying on biotechnology. By September 20,
to the content of plant drug extracted from around 232 centers of clinical tests were

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recorded, and in cases, even some reported result in destruction of useful germplasms of
their success in manufacturing the vaccine’s the medicinal plants. Modification and
phase I simultaneously. introduction of new types of medicinal plants
can be also time consuming and costly. Under
The inactivated or weakened biotic vaccines
these conditions, significant products with
can be quite effective in Covid 19 virus, but its
micro and limited market are always subjected
mass production and vast distribution were
to inattention. Besidesthese considerations, the
quite hard. Thesub-units of the vaccines can
fact that modern molecular methods used in
also be produced based on the recombinant
studying and improving medicinal types are
antigens such asS-protein/RBD (or VLPs
justifiable have caused specialists focus on
displaying said antigens) which are really
proposing new approaches which can be used
suitable formanufacturing and reproduction,
economically for a vast range of plant species.
and they can also be produced by molecular
To sum up, ongoing continuation of the modern
farming. The vaccine nvx-c0v2373which is
industrial life depends on paying more
produced based on the calculation obtained
attention to the long-term achievements of
from the spike clinical phase of recombinant
nature.
proteinshowed the immunity response and
tolerance of the antigen with a dose of 50 μg.
Conflict of interest
Therefore, it is injected in two 25- microgram
doses, that is, every 21 days [44]. None of the authors have any conflict of
interest to declare.
Neutral antibodies which act against corona
virus often break the relationship between the Consent for publications
group’s proteins and its receptors. In some
groups, S proteins are produced from poultry, All authors approved the final manuscript
pig, and rat corona viruses as well as the for publication.
interminable parts of Sprotein of SARS- CoV-2
produced successfully in the plants of corn, Availability of data and material
potato, tomato, and tobacco through Data are available on request from the
transferAgrobacterium with surface viruses of authors.
the plants. In all cases, the response of the
immunity system is represented after its oral Ethics approval and consent to participate
intake [45, 46]. However, transfer expression is
a quick method which in high quantities, is No human or animals were used in the
suitable for producing corona virus. In Italy, for present research.
example, there are two companies, Kentucky
BioProcessing (Owensboro, KY, United States) Acknowledgement
and iBio (Bryan, TX, United States) which We thank Payam e Noor University of Ilam for
produce subunits of the corona virus in their financial support.
accordance with the molecular farming.
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