This document discusses plant molecular pharming (PMP), which uses plants as bioreactors for producing recombinant pharmaceutical proteins. It covers the definition, history, strategies, host systems, production of antibiotics/enzymes/vaccines in plants, advantages/disadvantages of plant systems, and issues of transgene pollution. Key points include:
- PMP uses whole plants, plant cells or tissues to produce commercially valuable proteins like vaccines via recombinant DNA.
- Early work in 1986 produced human growth hormone in tobacco and sunflower. Commercial production of various proteins in plants has occurred.
- Strategies include transforming host plants, growing biomass, processing/purifying the product of interest.
- Plants,
this presentation deals with Molecular Ph(f)arming, and bio-safety issues related to it. This was presented by me in credit seminar in the division of Agricultural physics, IARI, New Delhi.
the sources used are duly acknowledged in the figures and slides.
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
this presentation deals with Molecular Ph(f)arming, and bio-safety issues related to it. This was presented by me in credit seminar in the division of Agricultural physics, IARI, New Delhi.
the sources used are duly acknowledged in the figures and slides.
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
This ppt explains about molecular farming, history of molecular farming, importance, basic process underlying it, its application in agriculture and its limitations
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
Vector mediated gene transfer methods for transgenesis in Plants.Akshay More
Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
This is about methods of creating transgenic animals,applications of transgenic animals in biotechnology and application of transgenic animals in pharmaceuticals.
This ppt explains about molecular farming, history of molecular farming, importance, basic process underlying it, its application in agriculture and its limitations
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
Vector mediated gene transfer methods for transgenesis in Plants.Akshay More
Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
This is about methods of creating transgenic animals,applications of transgenic animals in biotechnology and application of transgenic animals in pharmaceuticals.
It deals with application of such genes and proteins obtained from the animals especially for medicine and also industries. It is much useful to understand the basic.
Plant biopharming is defined as the farming of transgenic plants genetically modified to produce “humanised” pharmaceutical substances for use in humans.
Molecular insight into Gene Expression Using Digital RNAseq: Digital RNAseq W...QIAGEN
Gene expression profiling is the key to understanding biological pathways and complex cellular systems. In this webinar we will discuss the challenges of targeted RNA-seq data analysis and present the solutions provided by the QIAGEN automated online data analysis tools. Using raw sequencing data from targeted sequencing, the output of the QIAseq primary data analysis tool and the options in QIAseq secondary analysis, such as normalization strategies, will be described. The use of Ingenuity Pathway Analysis (IPA) to unlock the molecular insights buried in experimental data by quickly identifying relationships, mechanisms, functions, and pathways of relevance will be shown with an example.
Transgenic animal models for the functional analysis of vasoactive peptidesWaliullah Wali
The renin–angiotensin system (RAS) is a hormone system that regulates blood pressure and fluid balance.
renin–angiotensin system (RAS) is developed in animal by transgenic technology and effects of vasoactive peptide are seen.
Vasoactive peptide is a peptide hormone containing 28 amino acid residues.
Vasoactive peptide is produced in many tissues including the gut, pancreas, and suprachiasmatic nuclei of the hypothalamus in the brain.
It stimulates contractility of heart, causes vasodilation, lowers arterial blood pressure.
Vasoactive peptide has a half-life (t½) in the blood of about two minutes.
Transgenic technology has established to be very useful for the functional analysis of vasoactive peptide systems.
Chirality and its biological role (English language) - www.wespeakscience.comZeqir Kryeziu
This presentation focuses on organic chemistry, especially stereochemistry for 3D shape of molecules. When the same chemical substance differs in its spatial construct it changes in a drastic way its own features, in a biological environment.
Plant biotechnology also known as green biotechnology is the use of biotechnology in plant or crop production. There are several techniques used such as ell culturing. Organ culture, explant culture, cell suspension culture are some culture types. This is a very useful technology in which have several applications like synthetic seed production, somaclonal variation, cybridization, hybridization.
I have discussed Applications of Plant Tissue Culture under the following subheadings,
1. Micro Propagation
2. Clonal Propagation
3. Production of Genetically Variable Plants
4. Production of Virus Free Plants
5. Plant Breeding
6. Production of Useful Biochemicals
7. Preservation of Plant Genetic Resources
8. Importance of Tissue Culture in Biotechnology
Producing proteins or other metabolites useful to business or medicine in plants that are typically used in agriculture is known as molecular farming.
The practise of using plants to create recombinant protein products is known as molecular farming. The technology is now older than 30 years. The initial promise of molecular farming was predicated on three anticipated benefits: the low cost of plant cultivation, the enormous scalability of agricultural output, and the intrinsic safety of plants as hosts for the synthesis of medicines. As a result, a tonne of studies were published in which various proteins were expressed in various plant-based systems, and several businesses were established in an effort to commercialise the novel technology. For businesses making proteins for non-pharmaceutical uses, there was a modicum of success, but in the pharmaceutical industry, the hopes sparked by early, promising research were quickly dashed by the hard facts of industrial pragmatism.
Role of biotechnology in enhancing fruit crop production and qualityankit gawri
It was evident that developed biotechnological approaches have the potential to enhance the yield, quality, and shelf-life of fruits and vegetables to meet the demands of the 21st century. However, the developed biotech approaches for fruits and vegetables were more of academic jargon than a commercial reality
Genetically modified organisms (GMOs) are organisms in which the
genetic material has been altered using recombinant DNA technology.
Genetic manipulation involves a wide variety of modifications to produce
nutritionally valued GM crops. In some cases, genetic modifications
represent more faster and efficient mechanisms for achieving desired
resulting traits. This review indicate the mechanism of group of actions
with various biotechnological tool utilize to carry out genetic
modification, their benefits, etc. Production of GM food crops provides
new ways to fulfill future food requirments but risk associated factors
cannot be neglected. To overcome these problems and to cope with the
continuous increase in the number and variety of GMOs, new approaches
are needed. India has approved cultivation of some GM crops but due to
lack of proper knowledge and religious factors lead to stunted outcomes
ignoring environment cleanliness and hunger of malnourished segments.
So more attention still needed for its adoption globally by ensure its
safety for human utilization.
A transgenic crop plant contains a gene or genes which have been artificially inserted, instead of the plant acquiring them through pollination. The inserted gene sequence (known as the transgene) may come from another unrelated plant, or from a completely different species: for example, transgenic Bt corn, which produces its own insecticide, contains a gene from a bacterium. Plants containing transgenes are often called genetically modified or GM crops.
What is the need of transgenic plants?
A plant breeder tries to assemble a combination of genes in a crop plant which will make it as useful and productive as possible. The desirable genes may provide features such as higher yield or improved quality, pest or disease resistance, or tolerance to heat, cold and drought. This powerful tool enables plant breeders to do what they have always done - generate more useful and productive crop varieties containing new combinations of genes - but this approach expands the possibilities beyond the limitations imposed by traditional cross pollination and selection techniques.
Plant Genetic engineering ,Basic steps ,Advantages and disadvantagesTessaRaju
plant genetic engineering,first genetically engineered crop plant,first genetically engineered foods,genome editing,uses of GE,transgenic plants,basic process of plant genetic enginering,advantages and disadvantages of genetic engineering.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
Anti ulcer drugs and their Advance pharmacology ||
Anti-ulcer drugs are medications used to prevent and treat ulcers in the stomach and upper part of the small intestine (duodenal ulcers). These ulcers are often caused by an imbalance between stomach acid and the mucosal lining, which protects the stomach lining.
||Scope: Overview of various classes of anti-ulcer drugs, their mechanisms of action, indications, side effects, and clinical considerations.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
3. CONTENTS
• Definition
• History
• Molecular farming strategy
• Molecular farming host
• Plant molecular pharming
• Antibiotics, enzymes and vaccines produced from
microbes and plant
• Transgene pollution
• Case study
1/7/2017 3Dept. of Plant Biotechnology
4. DEFINITION
• The use of whole organisms, organs, tissues
or cells, or cell cultures, as bio-reactors for
the production of commercially valuable
products like recombinant proteins,
antibodies, vaccines via recombinant DNA
techniques.
• It is also known as Molecular farming or
Bio pharming.
1/7/2017 4Dept. of Plant Biotechnology
5. HISTORY
• 1986 - First plant -derived recombinant therapeutic protein-
human GH in tobacco & sunflower. (A. Barta, D. Thompson etal.)
• 1989 - First plant -derived recombinant antibody – full-sized IgG in
tobacco. (A. Hiatt, K. Bowdish)
• 1990 - First native human protein produced in plants –
human serum albumin in tobacco & potato. (P. C. Sijmons et al.)
• 1995 - First plant derived industrial enzyme – α-amylase in tobacco. (J.Pen,
L. Molendijk et al.)
1/7/2017 5Dept. of Plant Biotechnology
6. HISTORY
• 1986 First plant -derived recombinant therapeutic protein-
human GH in tobacco & sunflower. (A. Barta, D. Thompson et al.)
• 1997 First clinical trial using recombinant bacterial antigen
delivered in a transgenic potato. (C. O. Tacket et al.)
• 1997 Commercial production of avidin in maize.(E. E. Hood et al.)
• 2000 Human GH produced in tobacco chloroplast.(J. M. Staub et al.)
• 2003 Human GH produced in tobacco chloroplast.(J. M. Staub et al.).
Expression and assembly of a functional antibody in algae
Commercial production of bovine trypsin in maize.(S. L.
Woodard )
1/7/2017 6Dept. of Plant Biotechnology
7. 1. Clone a gene of interest
2. Transform the host platform species
3. Grow the host species, recover biomass
4. Process biomass
5. Purify product of interest
6. Deliver product of interest
Molecular Farming Strategy
1/7/2017 7Dept. of Plant Biotechnology
11. BACTERIA:
1. Do not produce glycosylated full –sized
antibodies.
2. Contaminating endotoxin difficult to remove.
3. Recombinant proteins often form inclusion
bodies.
4. Labour-and cost –intensive refolding in vitro
necessary.
5. Lower scalability
6. Preferred for the production of small,
aglycosylated proteins like Insulin, interferon-
β.
1. Limited by legal and ethical restriction
2. Require expensive equipment & media
3. Delicate nature of mammalian cells
4. Human pathogens and oncogenes
5. Scaling up problems
ANIMAL BASED SYSTEMS
1/7/2017 11Dept. of Plant Biotechnology
13. S. Biemelt;U. Sonnewald (2004)
Comparison Of Different Production Systems For Expression Of
Recombinant Proteins
1/7/2017 13Dept. of Plant Biotechnology
15. Plant Molecular Farming
1. Significantly lower production cost than with
transgenic animals, fermentation or bioreactors.
2. Infrastructure & expertise already exists for the
planting, harvesting & processing of plant material.
3. Plants contain no known human pathogens (such as
prions, virions,etc.) that could contaminate the final
product.
4. Higher plants generally synthesize proteins from
eukaryotes with correct folding, glycosylation &post
translational activity.
1/7/2017 15Dept. of Plant Biotechnology
16. 1. Plant cells can direct proteins to environments that
reduce degradation and therefore increase stability.
2. Low ethical concerns.
3. Easier purification (homologs don’t pose any
purification challenge, e.g.serum proteins or
antibodies).
4. Versatile(production of a broad diversity of proteins).
5. Take more time to develop.
6. Transgene & protein pollution.
1/7/2017 16Dept. of Plant Biotechnology
17. Expression systems for PMF
1. Transgenic plants
2. Plant -cell -suspension culture
3. Transplastomic plants
4. Transient expression system
5. Hydroponic cultures
1/7/2017 17Dept. of Plant Biotechnology
18. 1.Transgenic plants:
• Foreign DNA incorporated into the nuclear
genome using-
-Agrobacterium tumefaciens
-Particle bombardment
• Most common
• Long term non-refrigerated storage
• Scalability
• More ‘gene to protein’ time
• Biosafety concerns
1/7/2017 18Dept. of Plant Biotechnology
19. 2.Plant Cell Suspension Culture
1. Culture derived from
-transgenic explants
-Transformation after desegregation
2. Recombinant protein localization depends on –
-presence of targeting / leader peptides in the
-recombinant protein. Permeability of plant cell
wall for macromolecules
3. Containment & production under GMP procedure
4. Low scale up capacity
1/7/2017 19Dept. of Plant Biotechnology
21. 3.Transplastomic Plants:
1. DNA introduced into chloroplast genome
2. High transgene copy number
3. No gene silencing
4. Recombinant protein accumulate in chloroplast
5. Natural transgene containment
6. Long term storage not possible
7. Long development time
8. Limited use for production of therapeutic
glycoproteins
1/7/2017 21Dept. of Plant Biotechnology
22. 4.Transient expression system
1. Biolistic delivery of ‘naked DNA
• Usually reaches only a few cells
• Can be used for a rapid test for protein expression
2. Agroinfiltraion
•Delivery of Agrobacterium in intact leaf tissue by vacuum
infiltration
•Targets many more cells in a leaf
3. Infection with modified viralvector
1/7/2017 22Dept. of Plant Biotechnology
23. Virus Infected Plants
• Gene of interest is cloned into the genome of a
viral plant pathogen
• Infectious recombinant viral transcripts are
used to infect plants
• Rapid & systemic infection
• High level production soon after inoculation
• Genetic modification of plant is entirely
avoided
1/7/2017 23Dept. of Plant Biotechnology
25. 5.Hydroponic culture
• A signal peptide is attached to the recombinant protein
directing it to the secretory pathway
• Protein can be recovered from the root exudates
(Rhizosecretion) or leaf guttation fluid (Phylosecretion)
• Technology being developed by the US biotechnology
company Phytomedics Inc.
• Purification is easier
• Reduced fear of unintentional environmental release
• Expensive to operate hydroponic facilities
1/7/2017 25Dept. of Plant Biotechnology
26. Choice Of Host Species
Depends On:
• Protein To Be Produced & Its Desired
Application
• Transformation Efficiency
• Overall Production Cost
• Containment
1/7/2017 26Dept. of Plant Biotechnology
27. Comparison Of Various Plant
Expression Host Species
1/7/2017 27Dept. of Plant Biotechnology
35. Transgene Pollution –The Problems
•Transgene pollution is the spread of
transgenes beyond the intended genetically-
modified species by natural gene flow
mechanisms.
•Two classes of transgene pollution:
-The possible spread of primary
transgenes.
-The possible spread of superfluous DNA
sequences.
1/7/2017 35Dept. of Plant Biotechnology
36. Transgene Pollution –Possible Solutions
•Minimum required genetic modification.
•Elimination of non-essential genetic
information.
•Containment of essential transgenes.
•Alternative production systems transient
expression.
•Plant suspension cultures in sealed, sterile
reactor vessels
(Fischer et al., 1999a; Doran, 2000).
1/7/2017 36Dept. of Plant Biotechnology
37. 1. Use of lettuce, and viral vector-based transient expression systems to
develop a robust PMP production platform biological pharmaceutical agents
that is effective, safe, low-cost, and amenable to large-scale manufacturing
2. Geminiviral replicon system based on the bean yellow dwarf virus permits
high-level expression in lettuce of virus-like particles (VLP) derived from
the Norwalk virus capsid protein and therapeutic monoclonal antibodies
(mAbs) against Ebola and West Nile viruses.
1/7/2017 37Dept. of Plant Biotechnology
38. MATERIALS AND METHODS
1) Construction of expression vectors
• The construction of geminiviral vectors, pREP110,
pBYGFP, pBYNVCP, pBY-HL(6D8) Replicon and non-
replicon vector pP19 dual-replicon vector pBY-
HL(hE16).R
2) Lettuce agroinfiltration-
• Lettuce heads were vacuum infiltrated with GV3101
strains containing the targeted expression vectors
3) Protein extraction
• The crude leaf extract was processed by centrifugation at
to yield “lettuce extract”.
• Lettuce extract” was further clarified by filtration
through a 0.2 micron filter.1/7/2017 38Dept. of Plant Biotechnology
39. MATERIALS AND METHODS
4) Protein analysis-
• SDS-PAGE, Western blot, and ELISA analysis for
NVCP, 6D8 mAb and hE16 mAb,sucrose gradient
centrifugation and electron microscopy for NVCP VLP,
antigen binding assays for 6D8 and hE16 mAbs, and
GFP visualization were all performed.
5) WNV neutralization-
• The neutralizing activity of hE16 against WNV was
assessed using a focus reduction neutralization assay
6) Protein Purification-
• Anion exchange chromatography.
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40. RESULTS AND DISCUSSIONS
1. VISUALISATION OF GFP EXPRESSIONIN LETTUCE
Commercially produced lettuce heads were infiltrated with a single Agrobacterium culture, or co-
infiltrated with two or three cultures containing the indicated expression vector(s).
Leaves were examined and photographed 4 days post infiltration under UV (a–e) or regular light
(f).
Lettuce infiltrated with the infiltration buffer (a) was used as a negative control.
N. benthamiana was used as a positivecontrol (d). MagnICON vectors were described in
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41. 2.EXPRESSION OF NVCP IN LETTUCE LEAVES
Leaf protein extracts were separated on a 10% SDS-PAGE gel andtransferred onto
PVDF membranes probed with a rabbit polyclonal antibody against NVCP.
Lane 1: insect cell-derived NVCP standard;
lane 2: protein extract from uninfiltrated lettuce leaves (negative control); lane 3: extract from
pBYNVCP/pREP110 infiltrated lettuce leaves.
(b) Time course of NVCP expression-Total proteins from lettuce leaves infiltrated with
pBYNVCP/pREP110 or pBYNVCP/pREP110 + pP19
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42. 3.PURIFICATION AND CHARECTERISATION OF NVCP
Lane 1: Molecular weight marker;
Lane 2: insect cell-derived NVCP reference standard;
Lanes 3 and 4: crude protein extract and purified NVCP from N. benthamiana leaves as a
comparison;
Lane 5: crude extract from pBYNVCP/pREP110 infiltrated lettuce leaves; lane 6: purified NVCP
from lettuce leaves
b) Sucrose gradient sedimentation profile of purified NVCP. reference standard (I-NVCP)
c) Electron microscopy of lettuce-derived NVCP
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43. 4. EXPRESSION OF MAbS AGAINST EBV AND WNV
Total protein extracts of lettuce leaf were separated on 4–20% SDS-PAGE gradient gelstransferred to
PVDF membranes. The membranes were incubated with a goat
anti-human-gamma chain antibody to detect HC (a) or a goat anti-human-kappa chain
antibody to detect LC (b and c).
Lane 1: extract from uninfiltrated lettuce leaves;
lanes 2 and3: protein samples from lettuce infiltrated with pBY-HL(6D8).R or pBY-HL(hE16).R
construct;
lane 4: human IgG reference standard.
(d) ELISA analysis of 6D8 or hE16 mAb expression. Goat anti-human gamma and kappa chain antibodies
were used as capture and detection reagents, respectively to confirm the assembled forms of 6D8 or hE16
mAb
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44. 5. PURIFICATION OF MONOCLONAL ANTIBODIES
Lane 1: Molecular weight marker;
Lane 2: total leaf proteins from uninfiltrated lettuce leaves;
Lane 3: total leaf protein from lettuce leaves infiltrated with pBY HL(6D8).R;
Lane 4: purified 6D8 mAb
Lane 5: hE16 mAb purified from pBY-HL(hE16).1/7/2017 44Dept. of Plant Biotechnology
45. 6. CHARECTERIZATION OF MONOCLONALANTIBODIES
(a) Specific binding of 6D8 mAb to EBV. Tobaccoderived 6D8 (EBV T-6D8, positive
control), or a negative control generic human IgG.
(b) Binding of lettuce-derived hE16 to domain III of WNV E displayed on the cell surface of
yeast. Lettuce-produced hE16 mAb (L-hE16), mammalian cell-derived hE16 (M-hE16,
positive control), or a generic human IgG (h-IgG, negative control)
(c) Neutralization of WNV by lettuce-produced hE16 mAb. WNV was incubated with serial
dilutions of hE16 derived from lettuce (L-hE16) or mammalian cells (M-hE16) (positive
control) and used to infect Vero cells. Cells were then fixed, permeabilized, analyzed by
focus reduction assay and quantitated by Biospot analysis.1/7/2017 45Dept. of Plant Biotechnology
46. 1. BeYDV-based geminiviral replicon system can efficiently promote high-level
expression of NVCP VLP vaccine and anti-EBV or WNV mAb therapeutic
candidates in lettuce.
2. Using the geminiviral-lettuce system, the VLP andthe two therapeutic mAbs
accumulated to levels that were comparable to that observed in tobacco (Huang et
al., 2010; Lai et al., 2010), but higher than previously reported in lettuce using
non-viral vectors (Kapusta, 1999; Rosales-Mendoza et al., 2010; Webster et al.,
2006).
3. This procedures can efficiently isolate the NVCP vaccine candidate and the two
therapeutic mAbs to high (>95%) purity, in a scalable and cGMP compatible
format.
ANALYSIS AND CONCLUSION
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47. Perspectives on Molecular Pharming
• Use of virus infected plants is best approach for
molecular farming
• Molecular farming provides an opportunity for
the economical and large-scale production of
pharmaceuticals, industrial enzymes and technical
proteins that are currently produced at great
expense and in small quantities.
• We must ensure that these benefits are not
outweighed by risks to human health and the
environment
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48. References
• Robust production of virus-like particles and monoclonal
antibodies with geminiviral replicon vectors in
lettuce.Huafang Lai1, Junyun He1, Michael Engle2, Michael
S. Diamond2, and Qiang Chen1Plant Biotechnol J. 2012
January ; 10(1): 95–104. doi:10.1111/j.1467-
7652.2011.00649.x.
• Wikipedia
• (Rainer Fischer; Stefan Schillberg)
• Su-May Yu; Institute of Molecular Biology Academia Sinica
Nankang, Taipei
• S. Biemelt;U. Sonnewald (2004)
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