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CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No.

2 / 2010

Recuperado de: https://dialnet.unirioja.es/descarga/articulo/3717040.pdf

Free University - SECTIONAL PEREIRA

PROTEIN PLANTS AS FACTORIES recombinant human

Dora Janeth García J*

Artículo recibido: noviembre 15 de 2010 aprobado: diciembre 9 de 2010

PLANT MOLECULAR FARMING

Abstract

Plants have been used for centuries for medical purposes. Currently, plant
biotechnology has expanded the development of genetic engineering tools,
molecular biology, tissue culture and fermentation techniques have allowed the
growth of cells and organisms under controlled conditions, enabling the
production of materials for clinical and industrial potential. “Plant molecular
farming” is a new branch of biotechnology where the plants through genetic
engineering are modified to produce therapeutic proteins such as vaccines,
cytokines, antibodies, growth factors and enzymes while reducing contamination
risks, time and productions costs.

Keywords: Biotechnology, recombinants proteins, transgenic plants, plant


molecular farming, genetic engineer, Agrobacterium tumefaciens.

* Licenciada en Biología y Química. Candidata a magister en biología molecular y biotecnología. Junior Specialist in
Plant Transformation Research Center –PTRC-. Universidad de California Riverside. Correo Electrónico: dorag@ucr.edu
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

INTRODUCTION

The technology of the recombinant DNA and thegenetically modified


organism generation madepossible the expression of bovine human proteinsof ample pharm
aceutical value as and antibodiesin plants. These are known like recombinantproteins

The plants can be used like biorreactores for the protein production diverse. During several
diverse decades expression systems have been used for the recombinant protein production
including leavenings like Saccharomyces cerevisiae, transgenic microorganisms like
Escherichia coli, seaweed, cells and animal (especially of insects). Nevertheless, the plants
are a good model thanks to their low costs, stability of the protein, times of production, easy
to escalation and obtaining of the recombinant product.

The recombinant protein production implies the use of the biotechnology at diverse levels,
including methods of transformation, control of genetic expression, expression in diverse
platforms, selection of the protein to express, its accumulation, maintenance and stability. All
this is part of a new branch of the well-known biotechnology like “Plant molecular farming.

During many centuries the plants have provided us a molecule endless number with medical
and industrial applications. But, only as of years 80, with the production of the first
transgenic tobacco plants the beginnings of the technology of the DNA dreamed up
recombinante4 giving via the production in 1986 and 1989 of the human hormone of growth
and the first antibody in tobacco transgenic plants, respectively. These results showed that
the plants can assemble several functional multiprotein complexes, maintaining their
structural authenticity. In 1997 the first protein of high commercial value took place, the
avidina, expressed in transgenic maize. Since then the advantages and possibilities of the
technology have offered new opportunities for the production of an endless number of
composed of clinical and industrial importance.

A transgenic plant has been modified genetically with the incorporation in her genome of the
foreign gene that codifies a specific protein (for example, the gene that codifies collagen)
under the control of a constituent or induced promoter. This gene will produce the collagen
that later will be isolated of a specific organ of the plant. At the moment modified plants
genetically not only for use in feeding (as increasing their nutritional content in vitamins or
minerals) but also for the production of other compounds are generated as proteins or
diverse metabolites of pharmaceutical and/or industrial use with commercial potential like
enzymes, antibodies, sanguineous vaccines, hormones, proteins like albumens, cytokines,
diverse molecules of signaling, nutritional supplements and new polymers of clinical and
industrial interest.

The knowledge, relative facility and efficiency of transformation, the diminution of


environmental pollution hazards by flow genetic and easy to escalation does that plants like
tobacco, rice, soy, wheat, canola and barley are the main platforms used for the recombinant
protein production Nevertheless, other vegetal systems like Pope, carrot, lettuce, alfalfa,
arabidopsis and kidney bean also has been used for heterólogas protein expression. In this
revision are some aspects for the generation of transgenic plants, platforms of recombinant
protein expression and some expressed products of medical interest in plants, that at the
moment are in experimental phase or trade
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

GENETIC TRANSFORMATION IN PLANTS

The genetic engeneering is a tool used for the introduction of foreign genes (that can be of
the same or different species) in the plants and that provides advantages to them like the
resistance to insects, to viral diseases, fungoid or bacterial, tolerance to weed killers,
resistant plants to extreme conditions like alkalinity, salinity, drought, and cold. At the
moment the transgénesis in plants has ample applications, between which they stand out
the use of plants like genotoxicity sensors, environmental pollution and like biorreactores
used for the biological molecule production like antibodies and vaccines. The transgénesis
also is used in investigation to identify molecular, physiological, genetic and biochemical
aspects clearly of the plants. The genetic transformation is based on the DNA introduction
inside plant cells, for it requires a plasmídico vector in which insertóel interest gene, then,
this constructo genetic must finally be incorporated in the genome of the plant and the
totipotentes cells (present also in vegetal weaves) that have the capacity to be divided and
to regenerate a new seedling that will express or the characteristics of interest. (figura1) to
take to end a successful genetic transformation it is important to consider; constructo
genetic, the plasmídico vector, the type of promoter, the index of adaptation of the codon, an
efficient method for the introduction of the DNA in the genome of the plant cells and a good
system of vegetal regeneration that it allows the obtaining of new transgenic plants.

The transgénesis can be stable or transiente. The stable genetic transformation is that one
in which the incorporation of the foreign gene or interest occurs in the nuclear genome of the
plant and this information will be inherited to the following lineages. After several generations
the plants are only obtained homocigotas for the interest gene. The transference of the DNA
to the receiving cells was a process that began in years 60, but only years later and thanks
to events as the knowledge of the mechanism of formation of the nutgalls in plants by
Agrobacterium tumefaciens (negative Gram bacterium, pathogenic vegetable, able to
introduce DNA in the plant cells of natural form), 10 the discovery of the molecular
mechanism for the transference of the T-DNA to the genome of the plant and the cloning of
genes allowed the development of the vegetal genetic engeneering.

These facts opened the possibility of inducing new and diverse DNA segments (that codify a
specific protein) inside the plant cells generating in 1983 the first transgenic tobacco plants.

At the moment, the half-full genetic transformation by Agrobacterium is used in an ample


rank of vegetal species like mechanism of stable and transiente transformation. The
transformation of protoplasts using particle polietilenglicol or micro-injection and bombing are
other developed alternative methods that are also used depending the objective on the
transformation.
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

Although its efficiency is smaller, the transformation of plastidios is a method that has been
considered like an alternative strategy to the nuclear transformation. The cellular plastidios
are organelas that contain DNA, reason for which can carry out the incorporation of transgen
for the specific recombinant protein production. The plastidios have been considered
biocontenedores natural of transgen, are stable and ideal spaces to express high levels of
the protein in their interior. The selection of homoplastómicas plants (each chloroplast takes
transgen) is obtained after several generations from the regeneration of explantes of
bombed leaves, that have the capacity to be different themselves and to grow in selection
means. The biobalística is the method used for the transformation of organelas cellular like
the plastidios. The method involves the bombing of plant cells with gold covered microscopic
DNA particles.

The systems of transiente, ample transformation and are routinely used for the fast
validation of the constructos of expression. Within the methods of transiente expression it is
had:

1. Agroinfiltración; this method developed by Kapila15consists of the infiltration of to solution


of Agrobacterium tumefaciens (containing the T-DNA in the gene of interest) within the leaf
cells snuff facilitating the transfer of T-DNA to the cells.

2. Viral infection; This method is based on the ability of plant viruses to infect to plant.
Viruses such ace snuff mosaic (TMV) and potato X virus (PVX) have been used for ace
vectors the transfer of foreign genes into the plant. Using this expression system involves
the rapid processing of the recombinant protein to prevent degradation.
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

The recombinant protein production requires a system of stable expression of transgen,


since the method is based on the accumulation of the different protein of interest in
organelas vegetal, for example leaves, seeds, fruits, roots, plastidios. The location depends
on the system of expression, and the protein of interest.

Plant systems for the production of recombinant proteins

As superior eukaryotes, the presence of glycosylation systems and post-translational


mechanisms of plants, allow the synthesis of small peptides, polypeptides and functional
multiprotein complexes. In addition, plant cells present chaperones (proteins that help other
proteins in their folding, assembly and mobility within the cell) homologous to those present
in human cells facilitating the production, assembly and maintenance of the protein and,
thereby, facilitating A large diversity of biomolecules can potentially be produced in plant
systems.

Characteristics like the diminution of the pollution hazards by human pathogens, practicidad,
diminution of costs and times of production cause that the plants are considered like
invaluable and advisable on a large scale systems for the recombinant protein production
multiple. Factors like the amount of biomass, the recombinant by hectare, facility for
transformation and scaled protein production are considered when to select the platform for
the expression of a recombinant protein.

At the moment the seeds of some cereals like maize, rice, barley and wheat are considered
like good organs for the recombinant protein accumulation. These can be stored by long
periods and thanks to the phenolic compound absence it can maintain the stability of the
protein. In addition, the extraction of the end item from seeds is easy; sometimes fusions
have been made of the protein of interest to oily molecules that are associated to
membranes having allowed their fast separation of the vegetal system. And one of the main
advantages of the seeds is that the vaccine, antigen or pharmaceutical protein are provided
of oral form to be used like proteins for immunization, immunotherapy and treatment of
diseases.
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

Cellular Suspensions
The culture of plant cells in suspension is an alternative for the production of recombinant
proteins. This system requires in vitro sterile conditions, nutrient management and aeration
systems for the production of the protein of interest. One of the main advantages in the use
of this expression system is the reduction of protein heterogeneity due to the uniformity in
size and type of cells used. In addition, it is considered a rapid system since it does not
involve the regeneration and characterization of the transgenic plant, the costs are not high,
it is a safe method and productive cell lines can be generated in a few months allowing its
scaling. However, the decrease in the production of the protein in the stationary phase due
to the increase of proteolytic activity and the existence of few characterized cell lines are
limiting for this production platform. The Newcastle disease vaccine for chickens is an
example of a recombinant product expressed in suspension cells.

Recombinant protein produced in plants

Some expressed recombinant products in plants are in table 1.

Vaccines

A vaccine is an antigenic preparation that to the being applied in an organism allows the
immune system to respond and “to defend” before a particular disease. Animal bacteria,
leavenings, and cells like systems of recombinant vaccine expression have been used. One
of the most promising applications of the biorreactores plants as to produce vaccines is its
potential use for the production of vegetal weave antigens foods (bovine foods). The
antigenic protein production in vegetal weaves would allow to protect them of the
degradation in the gastrointestinal tract. Some vaccines expressed in tobacco, rice, maize,
soy, kidney bean, Pope, tomato and banana tree have been: Antigen of surface of the virus
of hepatitis B. B drive of the termolábil enterotoxina of Escherichia coli (LTB). B drive of the
toxin of Vibrio cholerae (CTB) the B-glycoprotein of human cytomegalovirus, the peptide D2
de Staphilococcus aureus, hemagglutinin, epítope of Plasmodium falciparum and protein G
of the virus of the rage. Some vaccines with veterinary intentions also have been expressed
in plants, for example for the avian influenza, the disease of Newcastle and hemagglutinin of
the expressed bovine plague in peanut.
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

Elección de la proteína de interés

Diseño y desarrollo del


Constructo genético

Selección de la plataforma
vegetal para la expresión de la
proteína

Transformación genética

Regeneración de la planta

Expresión de la proteína de
interés

Extracción y purificación del


producto recombinante

Evaluación clínica

Escalamiento y mercadeo

Figura 2. General scheme for the production of a recombinant protein in plants.


CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

Tabla 1. Some recombinant products derived from plants at the moment in clinical, commercial or experimental phase.

PLANTA PRODUCTO RECOMBINANTE COMPAÑÍA/ESTADO


Alazor Insulina SemBioSys Canadá
Alazor Inmunosphere SemBioSys Canadá
Alfalfa Colágeno Medicago Inc.
Arroz Hirudina (Anticoagulante) Applied Phytologies Inc.
Arroz Lactoferrina Ventria USA
Arroz Lisozima Humana Ventria USA
Arroz Factor Intrínseco Humano HIF Cobento Biotech AS- Dinamarca
Canola Hirudina SemBioSys Genetics Inc.
Cebada DERMOkine e ISOkine (Factores de ORF Genetics
crecimiento)
Espinaca Proteínas de fusión, epítopes de rabia Thomas Jefferson University
Lechuga Antígeno de superficie Hepatitis B Thomas Jefferson University
(HBsAg)
Maíz Avidina (Proteína de la leche) ProdiGene Inc.
Maíz Hirudina (Anticoagulante) Epicyte Pharmaceutical Inc.
Maíz Hirudina (Anticoagulante) IPT, Monsanto
Maíz Toxina Termo Lábil de E. coli ProdiGene USA
Maíz Proteína de la cápside del virus de la ProdiGene USA
gastroenteritis (TEGV)
Maíz Lipasa Gástrica Meristem Therapeutics France
Papa Proteína de la cápside del virus de Arizona State University
Norwalk
Papa Antígeno de superficie Hepatitis B Arizona State University
(HBsAg)
Papa Antígeno de superficie Hepatitis B AltaGen Bioscience Inc.
(HBsAg)
Tabaco Colágeno Meristem Therapeutics
Tabaco Colágeno Crop Tech Corp
Tabaco SEAP humana (Fosfatasa alcalina ) Large Scale Biology Corp.
Tabaco Inmunoglobulina G1 (IgG1) Epicyte Pharmaceutical Inc.
Tabaco Linfoma no Hodkings Large Scale Biology USA
Tabaco Antígeno Parvovirus felino Large Scale Biology USA
Tabaco Antígeno Papiloma Virus Large Scale Biology USA
Tabaco Proteína HN del virus de Newcastle Dow Agro Sciences
Tabaco Vacuna contra H5N1 Medicago USA
Tabaco Anticuerpo contra caries (CaroRX) Planet Biotechnology USA
Tabaco Terapia contra cáncer (DoxoRx) Planet Biotechnology USA
Tabaco Resfriado (RhinoRx) Planet Biotechnology USA
Tabaco IgG (ICAM1) Planet Biotechnology USA
Tabaco Anticuerpos contra Hepatitis B CIGB Cuba
Tabaco Alfa-Galactosidasa Planet Biotechnology USA
Tabaco
- Células en Proteínas HN del virus de Newcasttle Dow Agro Sciences
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

Recombinant antibodies

They are complex glicoprotéicos that recognize and they are united specifically to an
antigen. The individuality and specificity of union allow that the antibodies can be used in an
ample rank of applications like diagnosis, prevention and treatment of enfermedade. Some
expressed recombinant antibodies in tobacco, rice and arabidopsis are: IgG1 against the
carcinogenic antigen, employee in the therapy against the colon cancer and sine; antibody
mAb-so57 used as treatment for the virus of the rage; IgA/G-S1IgA/G against antigens of
surface of Streptococcus; Immunoglobuline To, G, DigA and s1gA against proteins of
cápside viral of the herpes simple virus, employee like inmunoprotección against genital
herpes; antibody m-Ab against human CD30, employee in the treatment of the lymphoma of
Hodgkin; MAK33IgG1 antibody against creatinin kinasa human MM used in mitocondriales
heart diseases, disorders, myopathies, miastenia and glycogen storage disease type V; Fab
fragment of MAK33 used in neuronal, rheumatic diseases and other that alter to the levels of
creatinin kinasa sérico; 38C13scFv used in the treatment of cell lymphoma.

Sanguineous proteins and other proteins and peptides of clinical use.

The sanguineous proteins are proteins found in blood plasma with diverse functions like:
The molecule transport diverse (lipids, hormones, vitamins and minerals); other proteins are
enzymes that participate in the cellular compound metabolism diverse and others are in
charge of the maintenance and regulation of the immune system. The sanguineous proteins
widely are used for diagnosis of diverse diseases, for example, albumen, produced
sanguineous protein in the liver, represent 50% of plasmatic proteins. Alteration in the
albumen normal energy levels can be associated to hepatic, renal or systemic disease.
Some expressed séricas proteins in plants are: albumen, aprotinina, encefalina, hemoglobin,
antitripsina α1. Other proteins with enzymatic activity expressed in plants are the protease
C, glucocerebrosidasa, human canine gastric lipase, acetilcolinesterasa and
transglutaminasa human. Some proteins and expressed peptides of clinical use in plants are
collagen, huridin, endostatin and lactoferrín, the stimulating factor of the macrophage,
interferon α, interferon β, interferon γ, hormone of human growth, erythropoietin, factor of
endothelial and interleukina vascular growth and factor of growth of interleukina.
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

Conclusión

The use of transgenic plants is widely used in recent years with high impact on human
health because of its great potential to be used as an alternative to increase the production
of vaccines, antibodies, proteins and various compounds of interest biopharmaceutical tool
large scale. The development of new promoters, development of new tools bioinformaticses,
the maintenance of the stability of the recombinant protein, the increase of the
transformation efficiency and the optimization of the extraction processes are new and
attractive centers of investigation. Their results will be translated in increases of production,
simplification of processes and in facility for the expression and production of a great
diversity of proteins or recombinant molecules that could be used for immunization,
diagnosis and/or treatment of multiple human diseases. The expression in weaves of
storage like seeds or tubercles in which the recombinant proteins can remain stable by long
periods to room temperature, will remove the necessity to establish and to maintain the cold
chain for the maintenance of the activity of the protein. This one of the great limitations for
the vaccine distribution in many regions of the world. In addition, the possibility of generating
vaccines foods will avoid the use of needles used in inmunoprotectores treatments; the
demand for the access and use will be increased significantly.

Gratefulness

To Martha Lucia Orozco, director of Plant Transformation Center - PTRC-University of


California Riverside by its authorization for the use of the photos of pages 1 and 3.
Transgenic plants of tomato growing in vitro in the middle of selection (Kanamicina 100mg/L)
and transgenic plants expressing the gene reporter GUS respectively.
CULTURA DEL CUIDADO ENFERMERÍA / VOL. 7 / No. 2 / 2010

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