Producción de Proteínas Farmacéuticas Recombinantes en Plantas (revisión)

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Information about Producción de Proteínas Farmacéuticas Recombinantes en Plantas (revisión)
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Published on March 11, 2014

Author: manuelgug

Source: slideshare.net

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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.

Manuel García Ulloa Gámiz

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.

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

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).

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.

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

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).

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

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.

• 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.

Transformation methods

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

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.

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

• 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.

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.

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

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