BIOTECHNOLOGY: THE EXPRESSION OF FOREIGN PROTEINS IN PLANTS

1
BIOTECHNOLOGY THE EXPRESSION OF FOREIGN
PROTEINS IN PLANTS
COURSE FIGURES CAN BE DOWNLOADED THROUGH
http//www.courseweb.uottawa.ca/BIO4174
2
BIOTECHNOLOGY Expression Systems

• Plants as the " factory" of choice!!
• Easily scaled up with standard agricultural
  practice
• Extensive infrastructure
• Economical approach for production and storage
• Safe with respect to human viruses or pathogens
• Wide range of host/expression systems
• both native and introduced

Course Map Plasmid vectors Viral
Vectors Expression Systems Escherichia coli
Other bacteria Yeast Pichia pastoris
Baculovirus Cell culture Plants Animals
3
BIOTECHNOLOGY Expression in Plants
But plants are not without potential problems for
the cloning of foreign proteins. 1. Need to
make fusions with host sequences such as
promoters 2.Post-translational modifications
such as protein processing and glycosylation may
differ form those of animal cells. 3. Insertion
is random (so far, see David Ow). Need for
extensive screening. 4. Long lead times
required for seed productions.
There are two general approaches to
production TRANSIENT INFECTION TRANSGENIC PLANTS
4
BIOTECHNOLOGY Expression in Plants
TRANSIENT SYSTEMS The basic idea is to use a
systemic plant virus to express the foreign
gene. As the virus spreads the amount of
protein increases. The virus itself is
disabled to avoid infection but uses
movement proteins to facilitate spread
between plant cells and tissues. Infection can
be done in a controlled environment.
5
BIOTECHNOLOGY Expression in Plants
Products include r-Aprotinin rh-Alpha
Galactosidase rh-Interferon Alpha 2a and 2b
non-Hodgkins lymphoma vaccines CaroRx,
antibody to control dental caries (with Planet
Biotechnology). http//www.lsbc.com/geneware.html
6
BIOTECHNOLOGY Expression in Plants
Use this technology to make antigens for vaccines
(VLPs) (Compare to M13 baculovirss technology
for Phage Display)
7
BIOTECHNOLOGY Expression in Plants
Advantages of transient technologies

• Rapid, can make vectors within weeks. No seed
  production.
• Scalable, no capital intensive manufacturing, Kg
  quantities of protein.
• Inherent physical and genetic control (eventually
  lose the ability to
• express the inserted gene).
• Flexible w.r.t. production and management.

But there are problems

• Requires constant high technology for a few
  species
• IP position blocks spread of technology (why not
  just do transformation
• with vesicles or naked DNA?)

But see Giritch et al (2006) Rapid high-yield
expression of full-size IgG antibodies in plants
coinfected with noncompeting viral vectors PNAS
103 14701-14706
8
BIOTECHNOLOGY Expression in Plants
TRANSGENIC SYSTEMS Transgenic systems are the
norm. We will focus on terrestrial plants but
keep in mind that transgenic algae have been
made. A few species have been extensively
exploited BUT many have been transformed. Many
plants have picked up foreign DNA.
9
BIOTECHNOLOGY Expression in Plants
There are two general methods of
transformation, Agrobacterium tumefaciens
infection and biolistics. Both follow a similar
pathway.

• Isolation and characterization of the gene
• Cloning of the gene into a suitable expression
  vector
• Transformation into plants or plant cells in
  culture
• Regeneration of plant.
• Establishment of transgenic lines for production
  (or breed
• into cultivars for agriculture)
• Isolation and purification of protein (if
  necessary)
• Formulation of protein product

10
BIOTECHNOLOGY Expression in Plants
There are two general methods of
transformation, Agrobacterium tumefaciens
infection and biolistics.
A.t infection, a natural process for many plants,
yeast and fungi
11
BIOTECHNOLOGY Expression in Plants
There are two general methods of
transformation, Agrobacterium tumefaciens
infection and biolistics.
Biolistics
12
BIOTECHNOLOGY Expression in Plants
Agrobacterium tumefaciens infection and
biolistics.
1.
4.
1. Inoculate (floral dip) 2. Growth of
explants on selective medium. 3. Regenerate
shoots and roots. 4. Regenerate plants for
testing and breeding.
3.
2.
13
Plant Expression Systems

• The Big Players are
• Single Cell Culture
• Foliage
• Root Extrusion Systems
• Seeds

14
BIOTECHNOLOGY Expression in Plants
Plant Expression Systems
SYSTEM Advantages Expression Single Cell
Culture Scale, Ease SC Protein of Growth,
Control Digitoxin(Digitalis lanata) Foliage,
Fruit Scale, Harvest or Ab, Vaccines,
etc Post-Harvest Root Extrusion
Continuous System GFP, xylanase System Ease of
purification Seeds Natural Protein Ab, etc.,
etc. Storage
15
MOLECULAR FARMING THE PRESENT
BIOTECHNOLOGY Expression in Plants

• Herbicide tolerant crops
• Roundup Ready Soybeans, corn, canola, cotton,
  etc.
• Glufosinate Soybeans, corn, canola, cotton,
  rice etc.
• Bt resistance crops
• Corn, cotton, tomato, canola, tobacco etc.
• Virus resistant crops
• Papaya

Why is there so much focus on a few crops??
See also http//www.colostate.edu/programs/lifes
ciences/TransgenicCrops/current.html
16
BIOTECHNOLOGY Expression in Plants
MOLECULAR FARMING, The Near Future

• Altered oil composition
• Vistive low linolenic soybean (2005)
• EPA in Brassica juncea
• Nutritional Modification
• Golden Rice with increased vitamin A
• high lysine corn for animal feed (2007)
• Increased or altered resistance to pests
  (bacteria, fungi etc.)
• Salt tolerant crops (salination)
• Plants for pharmaceuticals (MAbs, hormones,
  vaccines, etc.)

17
Lysine Maize Concept
BIOTECHNOLOGY Expression in Plants

• Animal feed is the largest use of US produced
  maize
• Conventional maize based diets for monogastrics
  contain less than optimal levels of some
  essential amino acids (e.g. lysine)
• Feed supplements ( complex or purified) are
  provided to improve dietary AA balance

• Objective
• To produce maize grain for animal feed use with
  increased available lysine content

maize utilization data (1996-1997)
This and the next 4 slides are from Monsanto
18
Lysine Maize
BIOTECHNOLOGY Expression in Plants

• Increase grain lysine content
• Conventional maize hybrids
• lt 0.01 free lysine
• 0.25 protein-bound lysine
• Target to increase 0.15 free lysine in grain
• 0.40 total lysine in grain
• 60 increase over conventional maize
• Targeting nutritional / typical dietary level
• Reduce or eliminate need for supplemental lysine

19
Lysine Maize
BIOTECHNOLOGY Expression in Plants

• Benefits
• Can reduce total dietary protein
• Can reduce nitrogen excretion
• Reduces need for synthetic lysine
• Decreased energy usage
• Reduced waste disposal

20
Approach to Increased Lysine
BIOTECHNOLOGY Expression in Plants
Deregulation of Diaminopimelate (Dap) pathway

• Aspartate ? ? ? Lysine
• main route of lysine synthesis in plants
• Dihydrodipicolinate synthase (mDHDPS/DapA) is a
  rate limiting step in lysine production in maize
• Lysine feedback inhibits mDHDPS
• ? flux through DHDPS
• ? lysine

21
Biochemical Basis of Lysine Maize
BIOTECHNOLOGY Expression in Plants

• Genetic Modification
• Introduction of lysine-insensitive
• variant of dihydrodipicolinate
• synthase (cDHDPS) from
• Corynebacterium glutamicum
• (CorDapA)
• Employ maize globulin1
• Promoter (predominantly
• maize germ expression)
• Desired Trait
• Accumulate lysine in
• the grain

22
BIOTECHNOLOGY Expression in Plants
GMO Rice

• Vitamin A deficiency is a major health problem

• Causes blindness
• Influences severity of diarrhea, measles

• gt100 million children suffer from the problem

• For many countries, the infrastructure doesnt
  exist
• to deliver vitamin pills

• Improved vitamin A content in widely consumed
  crops
• an attractive alternative

Phil McClean, Department of Plant Science North
Dakota State University
23
BIOTECHNOLOGY Expression in Plants
The Golden Rice Solution
?-Carotene Pathway Genes Added
Daffodil gene
Single bacterial gene performs both functions
Daffodil gene
24
BIOTECHNOLOGY Expression in Plants
The Golden Rice Solution
Golden Rice
Provitamin A content is increased from 1.6 µg/g
of rice to 37 µg/g of rice Al-Babili and
Beyer(2005) Trends in Plant Science 10(12)
566-573 Paine et al. (2006) Nature Biotech
23482-487 Provitamin A is enhanced in a wide
variety of crops Bai et al (2011) In Vitro Cell
Dev Biol 47 205-221
25
BIOTECHNOLOGY Expression in Plants
STRESS RESISTANCE
Water stress, drought tolerance and tolerance to
salts are major limitations to agriculture. The
problem is worsening due to competition for
water, drought, population increases and
salination of irrigation waters. GMOs can
help-we can select for plants with
increased tolerances to drought1, and salt2,3.
For several years CYMMIT has been breeding wheat
that is more pest resistant, and uses water more
efficiently anticipating a warmer dryer
climate.
26
BIOTECHNOLOGY Expression in Plants
Transgenic tomato plants overexpressing a
vacuolar Na/H antiport Top tomato
foliage Bottom Fruits A Wild type plant
in 5mM NaCl B Transgenic plant in 200mM NaCl
From Zhang, H-X. Blumwald, E .(2001).
Transgenic salt tolerant tomato plants
accumulate salt in the foliage but not in the
fruits. Nature Biotechnology 19765-768.
27
BIOTECHNOLOGY Expression in Plants
STRESS RESISTANCE
Transfer of 'HVAI1' (delayed leaf wilting) from
barley to wheat produces a drought-resistant
variety that requires only one-eighth as much
irrigation as conventional wheat. 3 years
of field trials beginning in 2001-2002. Can
be cultivated with rainfall alone in some
desert areas.
Bahieldina. A. et al. (2005) Field evaluation
of transgenic wheat plants stably expressing the
HVA1 gene for drought tolerance. Physiologia
Plantarum 123 421
28
BIOTECHNOLOGY Expression in Plants
Targeted Growth (http//www.targetedgrowth.com/)
The Company licensed two growth regulation
genes. Testing of the gene regulation effects
began in canola in 2003. Our second year of field
trials was carried out in 2004 at multiple sites
in Canada and the U.S. In both years of trials,
significant yield increases were
observed. Typical increases in crop yield
measure about 1 annually. Targeted Growth has
demonstrated average yearly increases of 10
12.
29
BIOTECHNOLOGY Expression in Plants
Expression in Plant Systems
From Ma et al. (2005) TIPS 10(12) 580-585
30
BIOTECHNOLOGY Expression in Plants
The rapid adoption of GMO plants in some
countries has been an amazing story. The
following few slides will illustrate.
31
BIOTECHNOLOGY Expression in Plants
32
BIOTECHNOLOGY Expression in Plants
33
BIOTECHNOLOGY Expression in Plants
34
(No Transcript)
35
PNTs approved to date (Canada)
BIOTECHNOLOGY Expression in Plants

• Agricultural Crops

• Traits
• Herbicide Tolerance
• Pest Resistance
• Nutritional Content

36
BIOTECHNOLOGY Expression in Plants
Environmental Safety Assessment Criteria

• Potential for PNT to become a weed of agriculture
  or be invasive of natural habitats
• Potential for gene-flow to wild relatives whose
  hybrid offspring may become more weedy/invasive
• Potential for the PNT to become a plant pest
• Potential impact of the PNT or its gene products
  on non-target species, including humans
• Potential impact on biodiversity

Why dont we apply these assessments to non-GMO
plants??
37
Safety Data Requirements for De-regulation of
Biotech Crops

• Commercialization 7 to 10 years -at least 9
  review stages
• Biotech crops and foods more thoroughly tested
  than conventional varieties ( assumed to be
  safe)- One biotech soybean subjected to 1,800
  separate analyses
• 23 feeding studies - dairy, beef, poultry,
  soy/corn equivalent in composition, digestibility
  and feeding value to non-GM. Clarke et al 2000
• Product description (7 items)
• Molecular characterization (17 items)
• Toxicity studies (as necessary) (5 items)
• Antibiotic resistance marker genes (4 items)
• Nutritional content (7 items)
• Substantial equivalence with parent variety
• Literature review and background
• Allergenicity potential
• Similarity to natural toxicants
• Anti-nutritional effects
• Protein digestibility
• Environmental aspects (5 items)
• Germination, growth, flowering studies (8 items)
• Ecological impact (5 items)

38
Relationship of the section Expression in
Plants to your the NSERC projects

• Can lead to higher accumulation of your protein
• Greenhouse vs field
• Trials and field trials
• Statistical analyses---YES!!