Article

Transgenic plants of Nicotiana tabacum L. express aglycosylated monoclonal antibody with antitumor activity

Abstract and Figures

The expression and production of pharmaceutical, industrial and veterinary proteins in plants is an attractive approach. These expression hosts bear an enormous production potential in terms of volume, easy processing of the starting material, and safety of the fi nal product due to the lack of pathogens able to infect animal and human cells. Antibodies are among the most frequently proteins expressed in plants, subsequently called plantibodies. However, plantibodies are differently glycosylated in plant cells, with oligosaccharide residues being added which may them immunogenic in the fi nal organism. For that reason, several strategies have been developed to genetically modify host plants to mimic the N-glycosylation patterns typical in animal cells. This work was aimed at developing a strategy to obtain a aglycosylated plantibody version of nimotuzumab, the fi rst antibody registered as a product in Cuba for cancer immunotherapy. The strategy comprised the genetic modifi cation of the heavy chain glycosylation site of nimotuzumab, and its expression as an aglycosylated protein in tobacco leaves, by means of developing a transient expression system using Agrobacterium infi ltration into tobacco leaves for the initial characterization of the plantibody. It was demonstrated that transgenic plants were capable of producing a plant-derived nimotuzumab antibody which retained the antitumor activity in vitro and in vivo, compared to its glycosylated counterpart produced in mammalian cells. This work demonstrates the potential of transgenic plants to produce aglycosylated therapeutic antibodies for cancer treatment, and won the National Award of the Academy of Sciences of Cuba in 2012.
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Corresponding author "
Transgenic plants of Nicotiana tabacum L. express
aglycosylated monoclonal antibody with antitumor activity
" Meilyn Rodríguez1, Merardo Pujol1, Lincidio Pérez1, Jorge V Gavilondo1,
Greta Garrido2, Marta Ayala1, Marlene Pérez1, Mónica Bequet-Romero1,
Gleysin Cabrera1, Osmani Ramos1, Ignacio Hernández3, Ernesto M González1,
Vivian Huerta1, Belinda Sánchez2, Cristina Mateo2, Ada Triguero1, Osmani Mendoza1,
Freya Freyre1, Carlos Borroto1
1 Centro de Ingeniería Genética y Biotecnología, CIGB
Ave. 31 e/ 158 y 190, Cubanacán, Playa, CP 11600, La Habana, Cuba
2 Centro de Inmunología Molecular, CIM
AP 16040, CP 11300, Playa, La Habana, Cuba
3 Centro de Isótopos, CENTIS
Ave. Monumental y Carretera “La Rada”, Km 31/2, Guanabacoa, La Habana, Cuba
E-mail: meilyn.rodriguez@cigb.edu.cu
ABSTRACT
The expression and production of pharmaceutical, industrial and veterinary proteins in plants is an attractive ap-
proach. These expression hosts bear an enormous production potential in terms of volume, easy processing of the
starting material, and safety of the nal product due to the lack of pathogens able to infect animal and human cells.
Antibodies are among the most frequently proteins expressed in plants, subsequently called plantibodies. However,
plantibodies are differently glycosylated in plant cells, with oligosaccharide residues being added which may them
immunogenic in the nal organism. For that reason, several strategies have been developed to genetically modify
host plants to mimic the N-glycosylation patterns typical in animal cells. This work was aimed at developing a strat-
egy to obtain a aglycosylated plantibody version of nimotuzumab, the rst antibody registered as a product in Cuba
for cancer immunotherapy. The strategy comprised the genetic modi cation of the heavy chain glycosylation site of
nimotuzumab, and its expression as an aglycosylated protein in tobacco leaves, by means of developing a transient
expression system using Agrobacterium in ltration into tobacco leaves for the initial characterization of the plantibody.
It was demonstrated that transgenic plants were capable of producing a plant-derived nimotuzumab antibody which
retained the antitumor activity in vitro and in vivo, compared to its glycosylated counterpart produced in mammalian
cells. This work demonstrates the potential of transgenic plants to produce aglycosylated therapeutic antibodies for
cancer treatment, and won the National Award of the Academy of Sciences of Cuba in 2012.
Keywords: molecular farming, plantibody, aglycosylation, epidermal growth factor receptor, nimotuzumab
Biotecnología Aplicada 2013;30:157-161
RESUMEN
Plantas transgénicas de Nicotiana tabacum L. expresan anticuerpo monoclonal aglicosilado con actividad
antitumoral. El empleo de plantas transgénicas para producir proteínas con nes farmacéuticos, industriales y ve-
terinarios es una estrategia promisoria. Este sistema hospedero tiene un gran potencial productivo, en términos de
volumen, fácil procesamiento y seguridad del producto nal, por la ausencia de patógenos que infecten las células
animales y humanas. Los anticuerpos están entre las proteínas más frecuentemente expresadas en las plantas, de-
nominados planticuerpos. Sin embargo, los planticuerpos se glicosilan diferencialmente en las plantas pues se les
adicionan residuos de oligosacáridos que pudieran hacerlos inmunogénicos en el organismo de destino. Con estos
nes se han implementado estrategias para modi car genéticamente las plantas de forma que reproduzcan los
patrones de N-glicosilación típicos de células animales. El propósito de este trabajo fue desarrollar una estrategia
para obtener una versión aglicosilada del anticuerpo nimotuzumab, el primero registrado en Cuba como producto
para la inmunoterapia del cáncer. Comprendió la modi cación genética del sitio de N-glicosilación en la cadena
pesada del anticuerpo y su expresión como proteína aglicosilada en hojas de tabaco. Para la caracterización inicial
del planticuerpo se desarrolló un sistema de expresión transitoria por in ltración de Agrobacterium en hojas de
tabaco. Las plantas transgénicas expresaron el planticuerpo PhR3 con similar actividad antitumoral que la mostrada
por el nimotuzumab producido en células de mamíferos. Este trabajo demuestra la potencialidad del sistema de
plantas transgénicas para producir anticuerpos terapéuticos aglicosilados para el tratamiento del cáncer, y mereció
el Premio Nacional de la Academia de Ciencias de Cuba en 2012.
Palabras clave: agricultura molecular, planticuerpo, aglicosilación, receptor de factor de crecimiento epidérmico,
nimotuzumab
Introduction
The use of plants as bioreactors, or molecular farm-
ing, is a technology comprising both, the expression
and characterization of recombinant proteins in plant
hosts and its high scale production (host plant cultiva-
tion, harvesting and biomass storage, processing and
puri cation of the protein of interest, and its related
REPORT
Meilyn Rodríguez et al. Report
158 Biotecnología Aplicada 2013; Vol.30, No.2
quality control processes and regulatory issues). The
use of plants as bioreactors is a relatively new ap-
proach for biopharmaceutical production.
A comparison of plant expression systems to
those based on other cell types shows that they have
the same ability to do most of the posttranslational
modi cations required for the proper conformation
of complex therapeutic proteins. Signi cantly, they
can be scaled up to biomass production volumes un-
reachable for any other expression systems, and has
the advantage of being generally-regarded-as-safe
hosts, unable to bear pathogens capable of infecting
animal and human cells. Noteworthy, it is possible to
directly administer biopharmaceuticals by oral route
as fruits, tubers, and others for therapy and vacci-
nation [1]. Plants as expression systems are highly
versatile, since they comprise various production
platforms: whole plants, seeds, cells in suspension,
roots, moss, algae and water duckweed. Furthermore,
the expression can be targeted to different subcellular
compartments or organs, by using speci c molecular
signals which facilitate better protection against post-
translational modi cations, including proteolysis [1].
Among the most frequent plant species can be found:
tobacco, tomato, banana, rice, corn, wheat, carrot,
soybean, potato, lettuce and alfalfa [1]. Tobacco
(Nicotiana tabacum L.) is the model by excellence
because of its easy manipulation and genetic trans-
formation, and its character of non-food crop which
minimizes the risk of contamination of the food chain
with recombinant proteins [2]. Particularly, tobacco
leaves-based expression provides additional bene ts,
such as, higher biomass yields and minimal leakage
of transformed genes into the environment due to the
elimination of owering; that is a shortcoming caused
by pollen or seed dispersal.
Nevertheless, and as for any other eukaryotic ex-
pression systems, plants are unable to exactly repro-
duce human-type glycosylation patterns on biophar-
maceuticals, taking into account that plant-speci c
glycosylation is considered at this stage as the major
limitation for the use of plant-made pharmaceuticals
in human therapy. To circumvent this disadvantage,
many groups have been working on the humanization
of protein N-glycosylation patterns by inactivating
plant endogenous and/or expressing heterologous gly-
cosyltransferases [3]. Another alternative comprises
universal glycan structures obtained by targeting the
recombinant protein to plant cell organelles with the
aid of several signals (i.e., the KDEL tetrapeptide, to
direct the expressed protein to the endoplasmic retic-
ulum, ER) [4]. Instead of the proteins being mainly
retained in those compartments, they also transit to
other subcellular organelles such as Golgi cisterns
[5], resulting in the addition of plant oligosaccharide
structures (sugar moieties such as β-(1,2)-xylose and
α-(1,3)-fucose). These processes may also vary, de-
pending on the given plant species.
Antibodies are the most common type of recom-
binant proteins expressed in plants (so-called planti-
bodies) [4], intended for human therapeutic applica-
tion in several human chronic diseases such as cancer,
autoimmunity and persistent infections [6] and with
real market perspectives. They are immunoglobulins
which may require a very speci c glycosylation pat-
tern for its therapeutic action, conditioning its proper
folding at the Fc region and for some subclasses of
human IgG, particularly IgG1, the activation of com-
plement system and antibody-dependent cell-mediat-
ed cytotoxicity (ADCC) [7]. At the same time, linked
glycans may also affect the stability, immunogenicity
and pharmacokinetic properties of these molecules.
There is still insuf cient information on whether
glycosylated plantibodies can be effectively used for
immunotherapeutics as their counterparts expressed
in mammalian cells, to activate the complement sys-
tem and promote ADCC. The same remains for the
possible glycosylation-associated immunogenicity
of the plantibody in humans, if it would be enough
to interfere with the plantibody therapeutic action.
Aglycosylation has also been investigated. Aglycosy-
lated plantibodies have been obtained by mutating the
N297 residue at the Fc region of the heavy chain, a
strategy successful for plantibodies whose native an-
tibody biological activity is independent of Fc region
effector functions [8], incapable of interacting with its
molecular target. As examples can be mentioned neu-
tralizing antibodies, agonists or antagonists, or even
when an active Fc fragment can produce unwanted
side effects [8].
Taking into account all these aspects, a strategy
was followed to obtain an aglycosylated plantibody
variant of the humanized monoclonal antibody
nimotuzumab (also known as hR3 or TheraCIM®),
a genetically engineered product from the Center
of Molecular Immunology (CIM, Havana, Cuba).
Nimotuzumab is an isotype IgG1 antibody which
targets the extracellular domain of the human epi-
dermal growth factor receptor (EGFR) and disrupts
the EGFR-associated signal transduction cascade
and mitogenic effects [9]. Some authors have report-
ed certain relationship between tumorigenicity and
EGFR overproduction in a variety of human tumors,
including lung cancer, astrocytic, head and neck
tumors, among others [10]. The chemical-pharma-
ceutical characterization of nimotuzumab, together
with preclinical information, production and testing
in clinical trials in Cuba, allowed the registration of
this product by the Cuban Regulatory Agency and
the Center for State Control of the Quality of Medi-
cines (Cecmed), as the rst therapeutic antibody for
the treatment of advanced head and neck in Cuba.
It has also been used in several countries, in more
than 10 therapeutic indications against several types
of tumors such as: colorectal, pancreatic, prostate,
esophageal and breast cancers [11].
The plantibody was obtained taking advantage of
previous works from several groups at the Center for
Genetic Engineering and Biotechnology (CIGB, Ha-
vana, Cuba), that established an ef cient platform for
the expression of heterologous proteins in plants [12].
The plantibody was expressed in transgenic Nicoti-
ana tabacum L. as an aglycosylated variant, obtained
by mutating the N-glycosylation site on the nimotu-
zumab heavy chain. The results supports the further
therapeutic evaluation of the plantibody regardless
the difference in glycosylation patterns depending on
the expression host.
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Meilyn Rodríguez et al. Report
159 Biotecnología Aplicada 2013; Vol.30, No.2
Results and discussion
Genetic construct for the expression in plants
of an aglycosylated nimotuzumab antibody
The heavy and light chains of the humanized nimotu-
zumab antibody were ampli ed by polymerase chain
reaction from the complementary DNA extracted
from a murine transfectoma producing the antibody
(donated by CIM). The design included suitable sites
for insertion of genes into the plant expression vector
(pHES74), and the plant endoplasmic reticulum sort-
ing amino acid signal (KDEL) at the carboxyl termi-
nus of both chains.
Additionally, the N297 amino acid was mutated to
Q in the antibody heavy chain, eliminating the single
glycosylation site of the immunoglobulin. Ampli ca-
tion products were sequenced, showing 98.2 % ho-
mology with the starting genes of the heavy and light
chains of nimotuzumab. The pHES74-based con-
structs bearing each antibody chain were sequentially
introduced into the binary vector pDE1001, resulting
in the pD-EGFR plasmid carrying both transcriptional
units in the same orientation. The tandem array of both
expression cassettes into a single T-DNA would favor
the coordinated expression of both genes. Thereafter,
Agrobacterium tumefaciens was transformed with the
plasmid of interest, for transient expression tests and
stable transformation of plants.
Expression of the aglycosylated antibody
in plant cells using a transient system
Our group developed a transient expression system
based on vacuum in ltration of A. tumefaciens into
the leaves of N. tabacum. This is a simple method by
which a bacterium is deposited into the leaf tissues
using a polycarbonate lter and, after infection, the
leaves are used to detect the expression of genes of in-
terest [13]. N. tabacum leaves in ltrated with A. tume-
faciens suspensions were harvested at different days,
while extraction of the total soluble proteins (TSP)
was performed for evaluation. Plantibody expression
was assessed by an ELISA detecting the presence of
human IgG, as shown in gure 1, for leaves harvested
on day 3 after infection.
Absorbance values obtained for the nimotuzumab
antibody (positive control) are 5 times higher than
the values used for the negative control of phosphate
buffer saline (B; Figure 1). The values in the case of
the TSP from leaves agroin ltrated with the A. tume-
faciens strain AT2260 (NC) were similar to those of
B. These results indicated that the analytical system
was speci c for the detection of human IgG and did
not react with other plant proteins. The optical den-
sity values of the TSP from leaves in ltrated with
AT2260::pD-EGFR (pD) were approximately 4-fold
those of the negative controls (Figure 1). These results
indicated that the testing system detects the antibody
produced in plant cells, and demonstrated the func-
tionality of the pD-EGFR construct to express the
aglycosylated variant of nimotuzumab.
The greater accumulation of the antibody detected
on day 5 after infection could be due to the stability
of the antibody within plant cells based on targeting to
the ER, and also to the conditions and functionality of
the tissue after agroinfection by the method developed
in this study. This system enabled us to produce 1.2 μg
of recombinant protein per gram of tissue, thereby fa-
cilitating downstream puri cation of the antibody by
protein A af nity chromatography. The puri ed prod-
uct was analyzed by SDS-PAGE and Western blot,
demonstrating the absence of plants glycans. The bio-
logical activity was con rmed by an indirect immu-
no uorescence assay using the human tumor cell line
A431 which over-expresses EGFR [13].
The implementation of agroinfection has advan-
tages over other microbial systems for plant transfor-
mation, because of allowing the proper expression and
postranslational processing of large heterologous com-
plex proteins. Another advantage comprised the lack
of sophisticated equipment and the low cost as com-
pared to other transient systems such as microinjec-
tion, microprojectile bombardment or electroporation.
Production of aglycosylated plantibody
from transgenic Nicotiana tabacum L. plants
Once the structural functionality and stability of the
plantibody were corroborated by the transient expres-
sion system, transgenic plants were obtained as stable
expression system able to generate progeny for pro-
duction purposes, either by sexual (seeds) or asexual
reproduction (by cuttings or micropropagation) [5].
Ninety-six transgenic plants were obtained by
genetic transformation of N. tabacum variety Ha-
vana 2.1.1 leaves with recombinant A. tumefaciens
AT2260::pD-EGFR. The presence of the antibody
was demonstrated in 90 % of the clones, with clone 86
showing the highest accumulation of the plantibody
(30 μg/g of fresh tissue). This plant line was propagat-
ed in vitro and subsequently grown under greenhouse
conditions to obtain a homozygous line.
Leaves from 6-to-8-weeks old clone 86 transgenic
plants were harvested and used as starting material to
develop a protein A af nity chromatography puri ca-
tion process. A 50 % recovery was obtained at 5 kg
scale, the plantibody preparation yielding 96 % pu-
rity as estimated by SDS-PAGE and gel densitometry.
Figure 1. Detection of human IgG by ELISA in transient expres-
sion assay. Samples represent 100 μg of total soluble protein
(TSP) analyzed at different dil; 1/20, 1/200 and 1/2000.
B: Extraction buffer, PBS 1× and 0.01 % Tween 20; NC: negati-
ve control, TSP from agroin ltrated leaves with Agrobacterium
tumefaciens strain AT2260; pD: TSP from agroin ltrated leaves
with AT2260::pD-EGFR construction; PC: positive control,
50 ng of nimotuzumab antibody. Bars stand for the mean plus
standard deviation values of optical densities of 5 experiments,
with 3 leaves analyzed in each case.
0.8
0.6
0.4
0.2
0
Optical density (405 nm)
Samples
dil 1/2000
B
dil 1/20
dil 1/200
NC pD PC
Rodriguez M, Ramirez NI, Ayala M, 13.
Freyre F, Perez L, Triguero A, et al. Transient
expression in tobacco leaves of an aglycosy-
lated recombinant antibody against the epi-
dermal growth factor receptor. Biotechnol
Bioeng. 2005;89(2):188-94.
Meilyn Rodríguez et al. Report
160 Biotecnología Aplicada 2013; Vol.30, No.2
The product was named PhR3 and subjected to further
analysis (Figure 2). Two bands of 25 and 50 kDa cor-
responding to the expected light and heavy chains of
PhR3 were observed in lanes of the eluates processed
under reducing conditions (Figure 2A). In the Western
blot assay under non-reducing conditions, PhR3 plan-
tibody appeared as a single band of high molecular
weight (approximately 150 kDa), comparable to that
of nimotuzumab (Figure 2B). The absence of oligo-
saccharides residues in the plantibody heavy chain
was corroborated by enzymatic digestion with PN-
Gase A followed by HPLC analysis [14].
Characterization of the immunochemical
properties, biological and antitumor activity
of the aglycosylated antibody
Puri ed PhR3 was characterized in vitro by using dif-
ferent techniques [14]. Its comparison to nimotuzumab
showed that despite slight differences, the aglycosy-
lation had no signi cant changes in the recognition
of EGFR by ELISA, in living cells ( ow cytometry
assay), nor on the ability of plantibody to inhibit
the phosphorylation and EGFR signaling. PhR3
was able to effectively displace radiolabeled EGF
in a competition binding assay for EGFR in the
microsomal fraction of human placenta. Also, the
Fab fragment of PhR3 had a 6.1 × 10-8 M dissocia-
tion constant, an af nity parameter similar to that
of nimotuzumab. In addition, PhR3 was also able to
block cell cycle progression in human tumor cells in
culture [14].
Moreover, similar results were found in the behavior
of both molecules when looking at possible in uences
of aglycosylation in pharmacokinetics and biodistribu-
tion of PhR3, and its anti-tumor effect in vivo (Fig-
ure 3). Both molecules had similar pharmacokinetic
PC 1 2 NC PC 1 2
150 kDa 150 kDa
25 kDa
0
1 4
20
A
18
16
14
12
10
8
6
4
2
Antibody concentration in serum (μg/mL)
Time (h)
0.50 8 12 16 20 24
hR3
PhR3
3000
2500
2000
800
600
400
200
0
B
Tumor volume (mm3)
Time (days)
5 10 15 20 25 30 35 400
PhR3 0.1 mg
PhR3 1.0 mg
hR3 0.1 mg
hR3 1.0 mg
PBS
Figure 3. Characterization of the PhR3 plantibody in vivo. A) Pharmacokinetic pro les of the PhR3 plantibody in Wistar rats. The curves represent the antibody concen-
trations. hR3: nimotuzumab-99mTc. PhR3: PhR3 plantibody-99mTc. Serum measurements, expressed as the average of 4 animals per group and the standard deviation.
B) Anti-tumor effect of PhR3 and nimotuzumab administered 24 h after A431 human tumor cell implantation in nude mice, at 0.1 or 1 mg/animal, for 10 days.
Phosphate-buffered saline (PBS) was used as negative control. Both antibodies exhibited a strong negative effect on tumor growth kinetics with respect to PBS. Data
are represented as volume mean values ± standard deviations.
Rodriguez M, Perez L, Gavilondo JV, 14.
Garrido G, Bequet-Romero M, Hernandez I,
et al. Comparative in vitro and experimental
in vivo studies of the anti-epidermal growth
factor receptor antibody nimotuzumab
and its aglycosylated form produced in
transgenic tobacco plants. Plant Biotechnol.
J. 2013;11(1):53-65.
Figure 2. Analysis of the purity and integrity of puri ed PhR3 plantibody. SDS-PAGE and Western blot of puri ed PhR3 plantibody.
A) Coomassie Blue-stained 12.5 % SDS-PAGE under reducing conditions; B) Western blot from samples transferred from 8 %
non-reducing SDS-PAGE, revealed with anti-human IgG-conjugated antibodies. PC: positive control, 10 μg of nimotuzumab
antibody; NC: negative control, total soluble protein (TSP) from non-transgenic plants; 1 and 2: eluates fractions from two
PhR3 plantibody preparations puri ed using a Protein A Sepharose IgG puri cation processes. Arrows indicate the molecular
sizes for each band.
Meilyn Rodríguez et al. Report
161 Biotecnología Aplicada 2013; Vol.30, No.2
pro les, characterized by a biphasic curve that could
beadjusted to a two compartment model (Figure 3A).
There was a statistically signi cant difference between
both antibodies with respect to the distribution phase
half-life (T½-α), indicating a larger and faster trans-
fer to the peripheral compartment for the PhR3 plan-
tibody. Noteworthy, no statistically signi cant differ-
ences were observed in the mean blood residence time
(MRT) between both antibodies.
Figure 3B shows the results of an assay performed
with different doses of both antibodies on nude mice
bearing human tumor xenografts. No signi cant dif-
ferences were found between groups of equal doses
and treatments neither with PhR3 nor nimotuzumab.
Conclusions
In summary, this work demonstrates that transgenic
plants can be successfully used to produce aglyco-
sylated versions of immunoglobulins intended for
therapeutic blocking of cell surface receptors or to
prevent its interaction with the soluble ligands. Agly-
cosylated plantibody variants can be equally active as
their mammalian counterparts, showing similar phar-
macokinetics and biodistribution and at the same time
exempt of plant-derived sugar moieties. Considering
that nimotuzumab has received several approvals as
therapeutic antibody for various speci c types of can-
cer, the plant-derived nimotuzumab antibody could be
a potential candidate for future cancer immunothera-
py clinical developments.
Relevance of the study
The main contributions of this work were: i) the im-
plementation of a strategy to express aglycosylated
antibodies in transgenic leaves of tobacco plants, by
using nimotuzumab as a model antibody; ii) the plant-
derived nimotuzumab antibody showed biological
properties similar to that of nimotuzumab, supporting
its potential therapeutic use [14]; and iii) the trans-
genic tobacco plants were able to produce aglycosy-
lated antibodies with the proper biological activity
[14, 15]. Methodologically, the study also provided a
new transient expression system to evaluate complex
molecules in plants [13].
Acknowledgements
The authors thank the following collaborators for
their contributions while conducting this study: Abel
Hernández, Osvaldo Oliva, Pedro Luis Ramos, Jeny
Soto, José Cremata and Kenia Tiel from the CIGB;
Rolando Pérez and Agustín Lage from CIM, and
Mariela León and René Leyva from CENTIS.
Pujol M, Gavilondo J, Ayala M, Rodriguez15.
M, Gonzalez EM, Perez L. Fighting cancer
with plant-expressed pharmaceuticals.
Trends Biotechnol. 2007;25(10):455-9.
... W opisywaną problematykę wpisują się też badania nad czynnikami terapeutycznymi w chorobach nowotworowych. I tak, w liściach N. tabacum uzyskano nimotuzumab -nieglikozykowane przeciwciała blokujące receptory epidermalnego czynnika wzrostu komórki [63]. Wykazano, że modyfikowane przeciwciała wykazują farmakokinetykę i biodystrybucję porównywalną z odpowiednikami pochodzącymi z komórek ssaków. ...
In recent years, there has been an increased interest of researchers in developing efficient plant heterologous expression systems of proteins for a wide range of applications. It represents an alternative to the traditional strategy utilizing bacterial, yeast, insect or mammalian cells. New techniques of identification and characterization and effective methods of plant genetic transformation allow the range of recombinant protein products to be expanded. Great expectations are associated with the use of plants as bioreactors for the production of specific proteins of therapeutic interest. This strategy offers a number of advantages, the most important being: the possibility of a significant reduction in production costs, the safety of the products obtained and full eukaryotic post-translational modifications of proteins. A group of proteins of special interest is pharmaceuticals, and a number of successful experiments have confirmed the possibility of obtaining heterogeneous proteins with therapeutic potential: monoclonal antibodies, vaccine antigens, and a variety of cytokines. This work is focused on selected recombinant proteins belonging to those groups expression of which was achieved in plant cells. These proteins may be used in the future for therapy or prevention of viral, bacterial or cancer diseases.
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