One of the main disadvantages of the production of recombinant proteins in transgenic plants by nuclear transformation is the low output level (generally less than 1% of the total soluble protein), which makes it difficult to establish large-scale industrial production.
The main advantage of plastidial transformation is that there are 5,000 - 10,000 copies of the genome in each plastid and so transgene expression is very efficient. Levels of recombinant protein of 47% of the total soluble protein have been achieved. Other additional advantages are: the incorporation of the transgene is produced by homologous recombination in a specific, known region thereby avoiding the position effect; genetic silencing does not occur; the possible dispersion of the transgene via pollen is avoided since pollen grains of the majority of cultivated species do not have plastids, and there are various systems for the elimination of the selection marker gene after the transformation.
Our laboratory has had several years of experience researching the production of different recombinant proteins in tobacco plants through chloroplastic transformation.
The promoters used are, amongst others: the psbA promoter (including the 5'UTR of the gene) and the Prrn promoter fused with the RBS of the G10L gene of the bacteriophage T7. Similarly, we have been working on the transfer of the available technology to commercial varieties of tobacco that produce high levels of biomass in order to maximise production of recombinant protein.
Human albumin (HSA) is one of the most widely used proteins in medicine. It is used in high doses in the treatment of patients with cirrhosis, shock, trauma and burns, amongst others. The cost of this treatment is very high. HSA is the most widely used intravenous protein in the world with an estimated global requirement of around 500 tonnes a year. The process of obtaining HSA through microbial expression systems has been studied. However, unlike the processing of smaller sized proteins, it is difficult to obtain high levels of HSA using this type of system. Using the culture of transgenic plants as a source of recombinant protein production, we have obtained HSA from potato tubers via nuclear transformation, with production levels of around 0.2% of the total soluble protein. However, through plastidial transformation we have managed to produce HSA in tobacco chloroplasts with average production levels of around 5% of the total protein. In this case the albumin is incorporated forming inclusion bodies.
The interferons (IFN) are a family of proteins known principally for their anti-viral properties. They are also potent immunomodulators and have antiproliferant properties. Consequently, IFNa is employed as the standard therapy for leukaemia, metastatic carcinomas, Karposi sarcoma, and hepatitis B or C. Despite the fact that IFNa2 is normally used, IFNa5 seems to be the form most often found in the liver and has as much anti-viral effect in hepatic tumoral cells as IFNa2. The interferon that is currently used comes from bacteria, but this system is incapable of providing sufficient IFNa to supply world need at a reasonable price. We are currently working on obtaining IFNa2 and IFNa5 from tobacco chloroplasts and have achieved IFNa2 expression levels of 4 - 8% of the total soluble protein.
The insulin-like growth factor 1 (IGF-1) is a potent anabolic hormone principally produced in the liver through stimulation by growth hormone. Patients with hepatic cirrhosis, muscular atrophy, osteopenia, and hypogonadism suffer from malnutrition that could be due to low levels of circulating IGF-1. In all these cases treatment with IGF-1 is recommended. Furthermore IGF-1 is also used in the treatment of many other illnesses. The enormous potential of IGF-1 has led many researchers to try to achieve its expression in various micro-organisms. The best results have been obtained in bacteria, which is how it is currently produced, despite the fact that the major part of the protein does not fold correctly which consequently makes its production more expensive and therefore treatment costs are very high. We are working on creating tobacco plants that express high levels of IGF-1 in their chloroplasts.
The cardiotrophin-1 (CTF1) is a protein from the interleukin-6 family that is related structurally and biologically with the cytokines that are found in heart, kidney, muscle and liver tissue. It was identified for the first time in 1995 through its ability to induce hypertrophia in immature cardiomyocytes and its ability to help the survival and proliferation of immature cardiomyocytes. Since then many other functions of CTF-1 have been described. It is therefore a relatively new substance but it is extremely promising for the treatment of a wide variety of diseases including severe hepatitis or hepatic damage caused by toxins. It could also be used to improve the condition of transplanted livers and to protect the remaining liver after resection of hepatic carcinomas. The product currently on the market is obtained from E. coli. We have recently also begun to express human CTF-1 from tobacco chloroplasts in our laboratory.
Since the beginning of the 90's different vaccine antigens have been produced from transgenic plants and it has been shown that they are capable of producing an immune response to important human or animal pathogens. Some of these are in the clinical studies phase and to give a concrete example, the vaccine against Norwalk disease is awaiting approval for commercial use in the USA. The production of vaccines from plants has the following advantages compared to traditional systems involving the administration of dead or attenuated viruses: greater biological security since plants do not become contaminated with human or animal pathogens; plant based vaccines can be administered in a simple form orally in the form of a single dose capsule, avoiding the use of needles and syringes; the vaccine does not have to be kept in refrigerated conditions; the production system is economical and can easily be put into large scale production using conventional agricultural techniques, and offers the possibility of producing multi-component vaccines.
In our group we are working with a canine parvovirus that produces haemorrhagic gastroenteritis in puppies and young animals. The 2L21 epitope corresponds to the N-terminal region of the VP2 protein of the viral capside and confers immunity when the synthetic peptide is administered parenterally. We have expressed in tobacco plants the 2L21 epitope fused with GFP (green fluorescent protein) or with CTB (cholera toxin sub-unit B). We have obtained levels of expression of the protein chimera of up to 31% of the total soluble protein, which is equivalent to 7.5mg/g fresh weight.
A mature plant at the moment of flowering can produce around 300mg of recombinant protein. The intraperitoneal immunisation of rats with crude extracts of total soluble protein led to a humoral immune response in the case of chimeral protein CTB-2L21 demonstrating that this protein is immunogenic but that the protein GFP-2L21 is not. This clarifies the stimulating role of the immune response of the CTB protein, transporting the epitope to the region where it triggers the immune response. We are currently carrying oral out immunisation trials and different doses of antigen.
We are also working in the production of a vaccine against human papillomavirus, directly responsible for cervical cancer. We chose as antigen L1 capsid protein of the virus for three reasons: to determine whether particles can form pseudovirales (VLPs) in chloroplasts, these VLPs could be used as a vaccine against cervical cancer, caused by the virus to study the ability of addressing and L1 protein interaction with antigen presenting cells (using a model of ovalbumin epitope), which activate cellular immune responses. They have obtained high levels of recombinant protein expression (24% of total soluble protein). Using electron microscopy and separation in sucrose gradients CLCS has verified the correct formation of VLPs in the chloroplast stroma. Intraperitoneal immunization of mice with plant extracts triggered a specific immune response with high titers of neutralizing antibodies were also.
In our laboratory we have cloned plastid thioredoxins f and m to snuff. Plants with overexpression of thioredoxin f showed an alteration in the accumulation of carbohydrates. Levels were observed up to 10 over 5 times higher starch and sucrose in leaf. These results were obtained with a non-commercial variety, Petite Havana, with low biomass production. Currently, this character is being transferred to commercial varieties. The calculations estimated production of starch from these varieties would be equivalent to improved crops such as barley or wheat.
Since cereals are currently used as feedstock for bioethanol production, the genetically improved snuff could be an alternative source of biomass in areas traditionally producing snuff (Extremadura, Andalusia). This will avoid using food crops used for biofuels production and output would be given a tobacco-growing areas of our country who see their futures in jeopardy by the disappearance of European aid to this crop.
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