High yield production of therapeutic proteins in chloroplast: By: Ravi Mehndiratta 2012BS24D High yield production of therapeutic proteins in chloroplast Introduction: Introduction Plastids of higher plants are semi-autonomous organelles with a small circular double stranded DNA with a high copy numbers and have their own transcription translation machinery. The chloroplast is one of the organelles known as plastids are found in plant cells and eukaryotic algae. Chloroplast are the primary source of the world’s food productivity as they are the site for photosynthesis. Chloroplast Transformation: Chloroplast Transformation The first chloroplast transformation was reported in Chlamydomonas using high velocity micro projectiles by biolistic delivery of naked DNA that integrated into the genome through homologous targeting (Boynton et al .,1988). The first stable plastid transformation was established soon in higher plants, Nicotiana tabacum ( (Daniell et al., 1990; Svab et al ., 1990). Up to date, plastid transformation has extended to many other higher plants, such as Arabidopsis ( Sikdar et al., 1998), rape (Hou et al., 2003), Lesquerella (Skarjinskaia et al., 2003), rice (Lee et al., 2006), potato ( Sidorov et al., 1999), lettuce ( Lelivelt et al., 2005), soybean (Dufourmantel et al., 2004), cotton (Kumar et al ., 2004a ), carrot (Kumar et al., 2004b), tomato ( Ruf et al ., 2001 ) and poplar (Okumura et al., 2006) Methods of Chloroplast Transformation: Methods of Chloroplast Transformation Two methods are currently available to stably transform plant plastids: the biolistic approach and the polyethylene glycol (PEG) treatment of protoplasts. Biolistic Method : plastid vector DNA is coated onto high-density tungsten or gold microprojectiles (0.6–1 μM diameter), which are then delivered at high velocity first through the cell wall and membrane, and then through the double-plastid membrane PEG Method : PEG Method Protoplasts are plant cells with their wall removed by enzyme treatment. Treatment of freshly isolated protoplasts with PEG allows permeabilization of the plasma membrane and facilitates uptake of DNA. Subsequently, with a mechanism largely uncharacterized, the plasmid DNA passes the plastid membranes and reaches the stroma where it integrates into the plastome as during biolistic transformation. A relatively small number of species have been transformed using this approach , mainly because it requires efficient isolation, culture and regeneration of protoplasts, a tedious and technically demanding in vitro technology. On the positive side, no special equipment is needed. Various steps in chloroplast genetic engineering: Various steps in chloroplast genetic engineering the recombinant DNA plasmid is bound to small gold nanoparticles that are then injected into the chloroplasts of a leaf using a “ gene gun” These plasmids contain multiple important genes: the therapeutic gene, a gene for antibiotic resistance , a gene that increases expression of the therapeutic gene, and two flanking sequences that ensure that the plasmid is not randomly integrated into the chloroplast genome(In brief, the flanking sequences guide the human recombinant DNA into a specific place on the chloroplast genome by binding to corresponding parts on the genome ) The leaf is then grown on a plate containing an antibiotic, which ensures that the only surviving plant cells will be those that contain the gene for antibiotic resistance and , therefore, contain the therapeutic gene as well These cells are then exposed to regenerative factors that induce them to start sprouting shoots and grow into full plants that express the desired protein Vectors: Vectors Initial transformation vectors carried a plastid 16S rRNA (rrn16 ) gene with point mutations that confer spectinomycin and streptomycin resistance. The recessive rrn16 marker genes were ,100-fold less efficient than the currently used aadA gene. The aadA gene encodes aminoglycoside 30-adenylyltransferase, an enzyme that inactivates spectinomycin and streptomycin by adenylation. Marker elimination system by CRE-loxP site specific recombination system: Marker elimination system by CRE- loxP site specific recombination system According to the CRE- loxP scheme, the marker gene (flanked by two directly oriented lox sites) and the gene of interest are introduced into the plastid genome in the absence of CRE activity. When elimination of the marker gene is required, a gene encoding a plastid-targeted CRE site-specific recombinase is introduced into the nucleus, excises sequences between the loxP sites. PowerPoint Presentation: Cre could be introduced by a second, Agrobacterium mediated transformation. The nuclear Cre is subsequently removed by segregation in the seed progeny. In tobacco, introduction of the nuclear Cre gene into the nucleus of transplastomic plants by Agrobacterium transformation extends the time needed to obtain marker-free plants by only one month. In an ideal case, it takes about six months to obtain a marker-free transplastomic tobacco plant that expresses a novel recombinant protein. Advantages of Chloroplast Transformation: Advantages of Chloroplast Transformation Since chloroplasts are almost always maternally transferred to the next progeny, there is little risk of any transgene flow from transplastomic plants to the neighboring weedy or wild relatives. Therefore, genes introduced into the chloroplast genomes can move only through seed, whereas the genes introduced into the nuclear genome can move through seed as well as pollen. possibility of expressing multiple genes in operons , high expression levels, possibility of expressing unmodified bacterial genes and human cDNA , and lack of gene silencing and position effects . The technology as such has numerous potent applications in developing plants resistant to biotic and abiotic stresses, and for production of therapeutic proteins and vaccines, which has already been demonstrated in the model plant system of tobacco, and potato and tomato. PowerPoint Presentation: A milestone in chloroplast engineering, the expression of Bacillus thuringiensis (Bt) Cry2Aa2 in transgenic chloroplasts resulted in high levels of accumulation (46.1% tsp), expression of a complete bacterial operon, and lack of the transgene product in pollen (De Cos et al., 2001).(Insect Resistance) Plastid transformation has also aided the study of plastid biogenesis and function. It has been used to investigate such areas as: plastid DNA replication origins promoter elements, RNA stability determinants, intron maturases , translation elements, protein import machinery, proteolysis, transgene movement and evolution, and transcription and translation of polycistrons ( Daniell , Cohill et al., 2004) AGRONOMIC TRAITS EXPRESSED VIA THE PLASTID GENOME: AGRONOMIC TRAITS EXPRESSED VIA THE PLASTID GENOME Many important agronomic traits have already been engineered via the plastid genome, such as herbicide resistance, insect resistance, and tolerance to drought and salt. Herbicide Resistance Herbicides are extensively used in agriculture, representing over $14 billion in use annually ( Kiely et al., 2004). The most commonly used herbicide, glyphosate , is a broad spectrum,nonselective , systemic herbicide. Glyphosate inhibits the enzyme 5-enolpyruvylshikimate- 3-phosphate synsthase (EPSPS), a nuclear-encoded gene involved in the biosynthesis of aromatic amino acids. PowerPoint Presentation: T ransgenic plants resistant to glyphosate are typically engineered to overexpress EPSPS. Because the target of glyphosate resides within the chloroplast, chloroplast transgenic engineering is an ideal strategy for developing glyphosate resistance in plants. Transgenic chloroplast plants were engineered to express a wild-type petunia EPSPS by integration of the aroA gene between the trnI and trnA genes in the inverted repeat region or rbcL and accD genes in the large single copy region ( Daniell et al., 1998). Homoplasmic integration of aroA was confirmed via Southern blot analysis and resulted in resistancto up to 5 mM glyphosate , 10-fold greater than the lethal concentration. PowerPoint Presentation: Drought Tolerance Trehalose phosphate synthase (encoded by the TPS1 gene) mediates the reaction that forms the osmoprotectant trehalose. Because trehalose has been found to accumulate under stress conditions such as freezing, heat, salt, or drought, it is thought to play a role in protecting cells against damage caused by these stresses. chloroplast transgenic plants grew normally and accumulated trehalose at levels 25-fold higher (Lee et al., 2003). Drought tolerance bioassays were performed in which transgenic and wild-type seeds were germinated in media containing 3 to 6 percent PEG. Chloroplast transgenic plants showed a high degree of drought tolerance by remaining green and healthy in 6 percent PEG, whereas wildtype plants were completely bleached Advantages of Chloroplast Biotechnology for production of therapeutic proteins: Advantages of Chloroplast Biotechnology for production of therapeutic proteins There are many advantages in producing human therapeutic proteins in plants. Plant systems provide opportunity for low cost production, the ability to carry out post-translational modifications and minimize the risk of contamination from potential human pathogens. Other advantages of plant derived therapeutic proteins include convenient storage, elimination of hospitals and health professionals for their delivery and the use of renewable resources for their production. The problem of gene silencing both at transcriptional and translational levels has not been observed in transgenic chloroplasts in spite of high levels of translation Production of High yield therapeutic proteins: Production of High yield therapeutic proteins The first plant-derived recombinant protein of pharmaceutical interest, the human growth hormone, was produced 25 years ago in tobacco and sunflower. Several bacterial antigens have been produced in plants using chloroplast transformation In the first report on the expression of an antigen in transplastomic plants, the B subunit of the cholera toxin (CTB) was expressed in tobacco chloroplasts from a dicistronic construct carrying the aadA gene and CTB regulated by the rrn promoter. PowerPoint Presentation: Chloroplast system is most suitable for high-level expression and economical production of therapeutic proteins in an environmentally friendly manner. However, the cost for purification of these proteins can be eliminated if they are orally delivered or minimized by the use of novel purification strategies. Oral delivery of therapeutic proteins is emerging as a new alternative for medical treatment and will benefit those who cannot afford the high cost of current treatments. Despite such rapid progress in the use of this organelle for plant molecular pharming , no glycoprotein has been expressed in transgenic chloroplasts, because N- or O- glycosylation is required for stability and functionality of many proteins. Chloroplast-derived therapeutic proteins: Chloroplast-derived therapeutic proteins Expression levels depend on the site of integration, regulatory elements used to enhance transcription/translation and the stability of the foreign protein. Genes coding for 20 amino acids or 83 kDa (PA) have been expressed in transgenic chloroplasts. Insulin like growth factor (IGF-1): Insulin like growth factor (IGF-1) Human insulin like growth factor (IGF-1) has therapeutic value not only in mediating the growth of muscle and other tissues, but its therapeutic value is being currently evaluated in diabetes, IGF-I induced neuroprotection, and in promoting bone healing. IGF-1 is a single-chain polypeptide with three disulfide bonds produced in the liver. PowerPoint Presentation: E. coli cannot produce the mature form of IGF-1, as disulfide bonds cannot be formed in E. coli cytoplasm. Since the IGF-1 gene has codons suitable for eukaryotic environment, codon optimization for chloroplast was performed to increase the levels of expression in transgenic tobacco plants. Surprisingly the expression levels achieved in transgenic tobacco were as high as 32% of the total soluble protein in both native and codon optimized genes. Human serum albumin (HSA): Human serum albumin (HSA) Human serum albumin (HSA), the most widely used intravenous protein, is obtained by fractionation of blood serum and accounts for about 60% of the total protein in the blood. When HSA was expressed in transgenic chloroplasts, the expression level obtained was up to 11.1% of the total soluble protein. This is 500-fold greater than the nuclear expression. Human interferon alpha (IFN2b): Human interferon alpha (IFN2b) Human interferon (IFN2b) is used in the treatment of malignant carcinoid tumors and has been shown to be very effective in the reduction of the tumor size. It also has other therapeutic values such as inhibition of viral replication, cell proliferation and enhancement of the immune response. PowerPoint Presentation: At present, this protein is produced in E. coli. It requires in vitro processing and purification. Interferon treatment is very expensive ($26,000–40,000 per treatment of hepatitis C). Nuclear expression of this protein resulted in very low expression levels (0.000017% fresh weight) in tobacco. When expressed in transgenic chloroplasts, the levels obtained were 18.1% of the total soluble protein. PowerPoint Presentation: The protein obtained was functional, as shown by its ability to protect HeLa cells against the cytopathic effect of encephalomyocarditis virus (EMC). This shows that chloroplast-derived IFN2b is just as active as commercially produced Intron A Antimicrobial peptide: Antimicrobial peptide Magainin is a broad-spectrum topical agent, a systemic antibiotic, a wound-healing stimulant, and an anticancer agent. A magainin analogue, MSI-99 was expressed in transgenic tobacco chloroplast and expression levels of approximately 21.5% of the total soluble protein were achieved. Pseudomonas aeruginosa , a gram-negative bacterium was used for testing the efficacy of the lytic peptide and this resulted in 96% growth inhibition of this pathogen. PowerPoint Presentation: Chloroplast-based expression of pharmaceuticals provides cost-effective benefits to the consumer. In order to establish the transplastomic biopharmaceuticals, the interferon α-2a gene along with aadA gene was flanked by the tobacco chloroplast inverted repeat region for two events of homologous recombination. Chloroplast transformation was accomplished upon bombardment of fully expanded 4 to 6 weeks-old tobacco leaves using helium gun. Green shoots regenerated from single antibiotic resistant cells were subjected to further rounds of selection and regeneration to develop homoplasmic clones. PowerPoint Presentation: The molecular analysis of the antibiotic-resistant plants confirms the presence of interferon alpha-2a as well as aadA genes in the plastid genome. The presence of a fragment of 20 kDa size on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography (HPLC) chromatogram confirms the expression of IFNA2a in the transgenic tobacco chloroplasts. MATERIALS AND METHODS: MATERIALS AND METHODS Plant material preparation for chloroplast transformation: Nicotiana tabacum was grown aseptically on 0.7% phyta -agar-solidified MS salts ( Murashige and Skoog , 1962), pH 5.8, containing 3% sucrose at 27°C under 100 μmol photons m–2 s–1 (16 h light, 8 h dark). Fully expanded leaves of plants 4 to 6 weeks old were used for chloroplast transformation. Vector construction for transplastomic biopharmaceuticals: Vector construction for transplastomic biopharmaceuticals Transplastomic vector was developed to express the synthetically engineered gene of high value pharmaceutical protein (Interferon alpha-2a) in tobacco plastome. The chloroplast transformation vector was comprised of left and right border sequences flanking the IFNA2 gene along with strong light inducible rbcL promoter that was cloned upstream to the antibiotic resistant gene aadA. The marker gene aadA was under the control of ribosomal RNA operon ( rrn ) promoter. The untranslated 3` region of psbA gene was used as terminator. PowerPoint Presentation: Selection and regeneration of transgenic plants. Molecular and protein analysis of transgenic plants PowerPoint Presentation: Limitations of Chloroplast Transformation 1. Transformation frequencies are much lower than those for nuclear genes. 2. Prolonged selection procedures under high selection pressure are required for the recovery of transformants . 3. The methods of transgene transfer into chloroplasts are limited, and they are either expensive or require regeneration from protoplasts. 4. These transformation systems are far more successful with tobacco than with other plant species. 5. Products of transgenes ordinarily accumulate in green parts only. Future of Plastid Biotechnology: Future of Plastid Biotechnology Transgene expression from the plant’s plastid genome has unique attractions to biotechnologists, including the plastids’ potential to accumulate foreign proteins to extraordinarily high levels and the increased biosafety provided by the maternal mode of plastid inheritance, which greatly reduces unwanted transgene transmission via pollen. To date, more than 50 different transgenes have been stably integrated and expressed via the plant plastid genome to confer important agronomic traits, as well as to produce industrially valuable biomaterials and therapeutic proteins. PowerPoint Presentation: Considering the recent scientific and technological developments in plastid transformation technology, such as the marker gene elimination systems, the possibility to induce gene expression, the development of novel purification method, and the selection of novel regulatory sequences for expression in chloroplasts, it can be predicted that in the next future the plastid transformation approach will be applied to a larger set of species and for a wider range of purposes.