Revisión del Artículo "Producción de Proteínas Farmacéuticas Recombinantes en Plantas" ("The Production of Recombinant Pharmaceutical Proteins in Plants"), de Julian K-C Ma, Pascal M. W. Drake y Paul Christou.

1. Manuel García Ulloa Gámiz

2. Recombinant Proteins
• Need for proteins:
research, medicine,
industry.
• A simple and
inexpensive system
that allows the large-
scale production of
safe recombinant
proteins would be
highly desirable.

3. • Traditional production systems that use microbial
fermentation, insect and mammalian cell cultures, and
transgenic animals have drawbacks:
– cost
– scalability
– product safety
– authenticity

4. Recombinant proteins expressed in plants
• 1986 – Human Growth Hormone (tobacco).
• 1989 – First Antibody (tobacco).
– Shows plants’ assemble capacity.
• 1992 – Hepatitis B Virus (HBV) Surface
Antigen.
– Confirms structural authenticity of plant derived
recombinant proteins (induced the expected
immune response after been injected into
mice).

5. Proteins as pharmaceuticals
Human Growth Hormone
(transplastomic tobacco)
– 7% of total soluble
protein.
Human Serum Albumine
(transplastomic tobacco)
– 11% of total soluble
protein.
Hirudin+Oleosin (canola)
– 0.3% of total soluble
protein.

6. Recombinant antibodies
•Yield = 1% of total
soluble protein
•Tobacco, cereal
seeds (provide
storage)

7. Recombinant subunit vaccines
• HBV vaccine.
• Oral vaccination.
– Potato and lettuce.
– **Protein requierements: structures that survive in the
extreme conditions of the human gut.
• Heat-labile toxin B subunit (LT-B) of ENTEROTOXIGENIC
Escherichia coli (ETEC) (0.3–10 mg produced high titres of
mucosal and systemic antibodies)
• Capsid protein of Norwalk virus (NVCP).

8. Other proteins of medical relevance
β-casein Lysozyme
Collagen + proline-4-
hydroxylase
Spider silk (Nephila
clavipes) (2% of the
total soluble protein).
Triple helix
self-assembly

9. Genetic aspects of molecular
farming in plants
• Promoters:
– Dicots: cauliflower mosaic virus (CaMV) promoter.
– Monocots: maize ubiquitin-1 promoter.
– Mechanical gene activation (MeGA) system: uses a
tomato hydroxy-3-methylglutaryl CoA reductase 2
(HMGR2) promoter, inducible by mechanical stress.
• Polyadenilation sites:
– CaMV 35S transcript.
– Agrobacterium tumefaciens nos gene.
– Pea ssu (ribosomal small subunit) gene.

10. • Secretory pathway is a more suitable
environment for folding and assembly than the
cytosol, leading to higher yields (antibodies).
– N-terminal SIGNAL PEPTIDE in the expression
construct.

11. Transformation methods

12. Post-translational modification
**α(1,3)fucose and
β(1,2)xylose
residues might
lead to adverse
reactions
a) disable the plant-
specific
fucosyltransferase and
xylosyltransferase
enzymes
b) modify plant-
derived
recombinant proteins
in vitro

13. Plant-expression hosts
• Tobacco production systems
– Can be cultured in homogeneous
suspension of single cells and small
clumps.
– The cultures can be maintained in
conventional microbial fermenters
with only minor technical
modifications.
– Product instability.
– Proteins that are larger than 20–30
kDa tend to be retained in the
apoplast.

14. • Cereals and legumes
– Long-term storage (3 years
at room temperature),
stability.
– Increased efficiency of
downstream processing.
– Nitrogen fixation.
– High ecological risk.

15. • Fruit and vegetables
(potato, tomato,
banana)
– Edible = suitable for
production of
recombinant subunit
vaccines, food additives
and antibodies for
topical passive
immunotherapy.
– Only works for specific
proteins.

16. Risks
• Transgene spread by pollen dispersal.
• Seed dispersal.
• Horizontal gene transfer.
• Effects of potentially toxic recombinant proteins on herbivores.
• Microorganisms in the rhizosphere and pollinating insects.
• Plant material that contains recombinant proteins could inadvertently enter the
food chain.

17. Conclusions
• PROS.
– Low cost of production.
– Rapid scalability.
– Absence of human pathogens.
– Ability to fold and assemble complex proteins accurately.
• TO DO LIST.
– Increase yields.
– Improve glycoprotein authenticity.
– Address biosafety and acceptability issues