Nov 2010
By analyzing the current situation in the protein market, we can see an increased demand for all types of industrial and medicinal proteins. For example, the monoclonal antibodies (mAbs), which are used in various clinical areas, represents the fastest growing sector in the biopharmaceutical market. We add to this list the enzymes (cellulase, α-amyalse), proteins that confer resistance to abiotic and biotic stresses, pharmaceuticals for oral delivery and parenteral administration and so on.
In this context, several plants system are used to produce recombinant proteins (r-proteins). Plants have several characteristics that recommend them for the production of r-proteins: the low cost of large-scale production, their ability to perform most post-translational maturation necessary for the biological activity of proteins synthesized and low risk for mammalian pathogen and other impurities contamination. However, using plants as bioreactors for the synthesis of proteins, raises a number of problems: the potential risks resulting from the release of the products of plants transgenic into the environment, the major structural differences between plant and mammalian N-linked glycans and the protein purification.
The synthesis of a particular type of r-protein in plants is proving to be a complex process because of the many elements involved. We continue to enumerate some characteristics of the biosynthesis plant process focusing on the transient expression systems.
So far, three approaches have been used to synthesize r-proteins in plant. Firstly, the genetic complement of the plant species is modified by the addition (insertion) the new genes into the plant chromosomes (stable transformation). Second, the expression from the plastid or mitochondrial genome. Transgenic plants are created by following methods: Agrobacterium-mediated transformation, polyethylene glycol-mediated transformation, liposome-mediated transformation, electroporation and particle bombardment. Finally, the expression due to the plant expression vectors (transient expression).
The methods of stable transformation used for the synthesis of r-proteins have many drawbacks. Among them: the transformation stages are laborious and slow, the dangers of horizontal recombinant genes transfer (pollen transfer, seeds and vegetative propagation), contamination of food and fodder plants because several food and feed crops are employed as production hosts. Under these conditions, the transient protein expression strategies may be a solution for r-protein synthesis. These methods have several advantages compared with traditional methods: speed and low cost of genetic manipulation and the quality of protein produced.
In review article ''Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems'', authors present two approaches of virus-based plant expression technologies: independent-virus (GENEWARE system, Kentucky BioProcessing ) and minimal-virus (magnlCON, Icon Genetics, Halle, Germany).
Independent virus systems have been derived from the genomes of several RNA-viruses (potexviruses - PVX, tabamoviruses - TMV and so on) and exploit systemic movement activities to infect host plants. By using the GENEWARE system have been produced: human enzymes, cytokines, subunit vaccine components and immunoglobulin fragments. GENEWARE expression system is a hybrid derived from tobacco mosaic virus (TMV). This system consists of T7 bacteriophage RNA promoter, replicase gene, two subgenomic promoters for the coat protein (CP) and movement protein (MP). R-aprotinin (serine protease inhibitor of bovine) was produced with good efficiency (cost and quality) using the expression GENEWARE system (3).
Minimal-virus systems (magnlCON system) is also derived from the genome of several RNA-viruses (PVX, TMV, TVCV and so on), but unlike independent virus system, the minimal-virus systems don't have the MP and CP genes. This transformation allows the production of larger proteins including cytokines, interferon, bacterial and viral antigens, growth hormone, single chain antibodies and monoclonal antibodies (mAbs) with good efficiency. Using this minimal expression system, Mapp Biopharmaceutical Inc produced an intravaginal antimicrobial agent (humanized αCCR5 mAb) to reduce mucosal transmission of HIV-1. In this case, the magnlCON system consists of two different virus expression vectors, TVCV and PVX, one for HC and another for LC of the αCCR5 mAb. The aerial portions of the plants (Nicotiana benthamiana) were harvested for extraction and purification of αCCR5 mAb (1.1 mg/ml) (3).
We must conclude that the use of the transient expression systems may be a solution for the production of r-proteins in non-genetically modified plants. For this remain to be solved some problems, such as: obtaining the ''humanized'' r-proteins in term of glycosylation (N-glycans), the potential risks resulting from the release of the products into the environment and to boost expression to ensure competitive production costs.
References
1. Daniell H. et al. (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends in Biotechnology. 23, 238-245.
2. Gomord V. et al. (2005) Biopharmaceutical production in plants: problems, solutions and opportunities. Trends Biotechnology. 23, 559-565.
3. Pogue GP. et al. (2010) Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems. Plant Biotechnology Journal. 8, 638-654.
4. Rademacher T. et al. (2008) Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains predominantly single-GlcNAc N-glycans. Plant Biotechnology Journal. 6, 189-201.
5. Sabharwal N. et al. (2007) Release of the recombinant proteins, human serum albumin, beta-glucuronidase, glycoprotein B from human cytomegalovirus, and green fluorescent protein, in root exudates from transgenic tobacco and their effects on microbes and enzymatic activities in soil. Plant Physiology and Biochemistry. 45, 464-469.
6. Streatfield SJ. (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnology Journal. 5, 2-15.
