Comparison of features of recombinant protein production in existing systems (according to Fischer and Emans 2004; worked out /modified on the basis of Demain and Vaishnav 2009).
1. Introduction
Recombinant proteins can be expressed in transformed cell cultures of bacteria, yeasts, molds, mammals, plants, insects, or via transgenic plants and animals. Numerous factors influence quality, functionality, yield and protein production rate, so the choice of appropriate expression system is of primary importance. During last few years, plants have become an increasingly promising and attractive platform for recombinant protein production (Basaran & Rodriguez–Cerezo, 2008). Progress in recombinant DNA technology, plant transformation and
Other advantages of the plant–based expression systems include: high scalability (in the case of field cultivation), low production cost of biomass (agriculture), in some cases low upstream costs (edible vaccines, purification process can be omitted), and what is most important - the ability to produce target proteins with desired structures and biological functions (Boehm, 2007). Recombinant proteins expressed in plants can be accumulated to a high level in seed endosperm, fruit or storage organs (e.g. tubers, roots) or secreted directly to the culture media. Because plant culture media contain no exogenous proteins, the recovery of recombinant proteins from a medium is expected to be much simpler and less expensive than the recovery from homogenized biomass (Cox et al., 2009).
Features Transgenic plants Plants viruses Yeast Bacteria Mammalian cell culture Transgenic animals Cost/storage Cheap Cheap Cheap Cheap Expensive Expensive Distribution Easy Easy Feasible Feasible Difficult Difficult Gene size Not limited Limited Unknown Unknown Limited Limited Glycosylation Correct Correct Incorrect Absent Correct Correct Production costs Low Low Medium Medium High High Production scale Worldwide Worldwide Limited Limited Limited Limited Propagation Easy Feasible Easy Easy Hard Feasible Protein folding accuracy High High Medium Low High High Protein homogeneity High Medium Medium Low Medium Low Protein yield High Very high High Medium Medium-high High Safety High High Unknown Low Medium High Scale up costs Low Low High High High High Therapeutic risk Unknown Unknown Unknown Yes Yes Yes Time required Medium Low Medium Low High High |
Transgenic plants | Plants viruses | Yeast | Bacteria | Mammalian cell culture | Transgenic animals |
Cost/storage | Cheap | Cheap | Cheap | Cheap | Expensive | Expensive |
Distribution | Easy | Easy | Feasible | Feasible | Difficult | Difficult |
Gene size | Not limited | Limited | Unknown | Unknown | Limited | Limited |
Glycosylation | Correct | Correct | Incorrect | Absent | Correct | Correct |
Production costs | Low | Low | Medium | Medium | High | High |
Production scale | Worldwide | Worldwide | Limited | Limited | Limited | Limited |
Propagation | Easy | Feasible | Easy | Easy | Hard | Feasible |
Protein folding accuracy | High | High | Medium | Low | High | High |
Protein homogeneity | High | Medium | Medium | Low | Medium | Low |
Protein yield | High | Very high | High | Medium | Medium-high | High |
Safety | High | High | Unknown | Low | Medium | High |
Scale up costs | Low | Low | High | High | High | High |
Therapeutic risk | Unknown | Unknown | Unknown | Yes | Yes | Yes |
Time required | Medium | Low | Medium | Low | High | High |
The usage of aquatic plants e.g.
Feasible storage of recombinant proteins in desiccated plant parts excludes the requirement for its immediate isolation and lowers the risk of the loss of biological function during prolonged freezing of preparations. For example, antibodies or vaccines expressed in cereal seeds remain stable at ambient temperatures for years (Stoger et al., 2002). Until recently, low accumulation levels have been the major bottleneck for plant-made recombinant protein production. However, several breakthroughs have been done during past few years allowing for high accumulation levels. Mainly through chloroplast, vacuole, ER lumen transient expression, coupled with subcellular targeting and protein fusions (Sharma and Sharma, 2009). Viral transfection and agroinfiltration are promising alternative strategies ensuring increase in yields and speeding up the development of an expression platform (Gleba et al., 2005). On the other hand, plant–based expression systems are different from the mammalian host pattern of glycosylation. The occurrence has raised concerns regarding the potential immunogenicity of plant-specific complex N-glycans ( α 1,3-fucose and β 1,2-xylose residue), which are present in the heavy chains of plant-derived antibodies (Gomord and Faye 2004). The above mentioned residues have been confirmed not only to induce immune response but also to make foreign proteins undergo a conformational change making them different from the native ones which results in decrease in their biological activity. However, some achievements in humanized glycosylation or removal of enzymatic pathway generating immunogenic residues on glycoproteins have been reported. Recently it has been shown that glycoengineered moss (
Other approaches to overcome undesirable glycosylation accommodate export of foreign proteins into subcellular compartments: ER lumen, where glycosylation characteristic of plants does not take place; cytosol, where glycosylation process is not found; or recombinant protein expression export into plastids (proteins do not undergo glycosylation there). According to several studies ER targeting gives higher yield of biologically active protein than cytosol targeting (referred by Boehm, 2007).
Potential disadvantages of transgenic plants include possible contamination with pesticides, herbicides, and toxic plant metabolites. Proteolytic degradation, post/transcriptional gene silencing, position effect and transgenic recombination are other obstacles affecting stability or expression level of transgenic plants (Basaran and Rodriguez–Cerezo, 2008).
The public concern about health and environmental risk associated with transgenic plants is being considered at different levels: inherent risk of transgene leakage into non-transgene crops or naturally occurring wild type species (transgene escape through pollen); transgene spread by seed or fruit dispersal; horizontal gene transfer by asexual means; unintentional exposure of non-targeted organisms (e.g. birds, insects or soil microorganism); elicitation of allergic response/reaction in people (Basaran and Rodriguez–Cerezo, 2008). There are some strategies which allow to alleviate these problems including usage of closed culture facilities, such as greenhouses, hydroponic or suspension bioreactors or plastid transformation (as plastids are inherited through maternal tissues in most species and the pollen does not contain chloroplasts, hence the transgene cannot be transferred) (Basaran and Rodriguez–Cerezo, 2008).
From economical point of view, plants can be an alternative system for recombinant protein production (especially biopharmaceutical) in comparison to those exploiting mammalian or bacterial cell cultures. In this system a desired foreign protein can be produced at 2-10% of the cost of microbial fermentation system and at 0.1% of mammalian cell cultures, although it depends on the protein of interest, product field and a plant used. In general, the recombinant protein yields up to 1.5% of the total soluble protein (TSP). For example the content of antibodies does not exceed 0.35%-2% and vaccines- 0.01-0.4% of TSP (Basaran and Rodriguez–Cerezo, 2008). On the other hand, phytase from
2. Expression strategies
Gene expression and synthesis of proteins is a complex multi-step process. For efficient expression of recombinant proteins in plants, it is essential to optimize every step of the process for the plant machinery. This includes the methods of plant transformation, the choice of a transgene promoter, improvement of transcript stability and the efficiency of its translation. After translation, the protein needs to be accumulated in plant cells or effectively secreted.
2.1 Stable nuclear transformation
The first step in plant transformation consists in the entrance of a desired genomic sequence into a plant cell. Stable nuclear transformation is caused by integration of the recombinant DNA in the nuclear genome. DNA can be transferred into the nuclear genome by either direct (e.g. biolistics) or indirect (e.g.
In the stable nuclear transformation whole plants can be regenerated, eventually producing a seed stock or a plant tissue maintained in an aseptic culture. The advantage of this system is that the transgene is heritable, permitting the establishment of a seed stock for future use. Establishment and characterization of stable transgenic lines can be costly and time consuming. Large numbers of transgenic lines need to be screened and analyzed before a single optimal line can be selected for protein production (Ling et al., 2010). Other disadvantages are gene silencing and position effects.
Nuclear transformation has been employed and extensively studied in many plant species, however, it generally results in low expression of soluble foreign proteins (Yap & Smith, 2010).
Recombinant proteins can be targeted to different subcellular compartments in plant cells, such as cytostol, apoplast, endoplasmic reticulum, vacuole or chloroplast.
2.2. Transplastomics
Using particle bombardment or polyethylene glycol (PEG) treatment, DNA can be targeted into the chloroplast genome (Yusibov & Rabindran, 2008). Each cell contains a large number of plastids, ~100 chloroplasts per cell, and each of them contains about 100 genomes. Transplastomic lines vs. nuclear ones have significantly greater yield of foreign proteins (1-20% TSP) due to the high number of copies of the chloroplast genome and they offer major advantage in terms of transgene containment, as chloroplast genomes are predominantly maternally inherited, limiting out-crossing of the transgenic pollen. No transcriptional or post-transcriptional silencing effects have been observed in chloroplast transformation (Yap & Smith, 2010). Chloroplasts also support operon based on transgene allowing the expression of multiple proteins from a single transcript. There are two disadvantages of the chloroplast system – first: chloroplast transformation is not a standard procedure and is thus far limited to a relatively small number of crops, second: lack of some of the eukaryotic machinery for post-translational modification (Yusibov & Rabindran, 2008).
Gene integration in the plastid genome occurs by means of two homologous recombinant events mediated by a bacterial-like Rec A based system. Vectors include two ‘targeting’ regions flanking the selectable marker gene and a cloning site for insertion of the gene of interest. The targeting regions are between 1 and 2 kb in size and are plastid DNA sequences able to direct transgenic integration into plastome intergenic regions. Integration by homologues recombination in a preselected genome region enables insertion of only transgenic sequences and prevents uncontrollable variation in the expression of transgene. Strong promoters for plastid encoded polymerase (PEP) from the
2.3. Optimization of expression level
Increasing the transcription rate of stably transformed gene sequences is the most direct and efficient approach to increase protein expression. This is mainly achieved with the use of a strong constitutive or inducible promoter. Constitutive promoters directly drive the expression in all plant tissues and are independent of the production host developmental stage. The best known and most widely used constitutive promoter in plant biotechnology is derived from
Inducible promoters allow external regulation by chemical stimuli such as alcohol, steroids, salts, sucrose or environmental factors such as temperature, light, oxidative stress and wounding. Inducible expression is advantageous as this allows protein production to be separated from cell growth. The use of chemical inducible promoters in combination with the chemical responsive transcription factor can further restrict the target transgene expression to specific organs, tissues or even cell types (Zuo & Chua, 2000). The examples of inducible promoters and synthetic transcription activators are: the rice α-amylase 3D (
Tissue-specific promoters control gene expression in a tissue or in a developmental stage specific way. The transgen driven by such a promoter is expressed in a specific tissue leaving all the other tissues unaffected. It helps to force transgene expression in storage organs like seeds, tubers or fruits. Several of such promoters were tested: tuber specific patatin promoter (Jefferson et al., 1990), fruit specific E8 promoter (Jiang et al., 2007), arcelin promoter (Osborn et al., 1988), maize globulin 1 promoter (Rusell & Fromm, 1997), 7s globulin promoter (Fogher, 2000), rice glutelin promoter (Wu et al., 1988) and soybean P-conglycinin subunit promoter (Chen et al., 1986).
The optimization of promoters activity can be further improved by means of engineered DNA elements - enhancers, activators or repressors located up or downstream of the core promoter. Enhancers are shown to increase gene expression when placed proximally to the promoter, they bind activator proteins and promote RNA polymerase II placement at the TATA box. Transcription is also enhanced with flanking the transgene by nuclear scaffold/matrix attachment regions (S/MARs) important for structural organization of eukaryotic chromatin (Halweg et al., 2005).
The translational efficiency of a transgene is determined by proper processing (capping, splicing, polyadenylation, nuclear export) and mRNA stability. The 5’ and 3’ untranslated region (UTR) of the plant mRNA plays crucial roles in its processing (Cowen et al., 2007). The 5’-UTR is very important for 5’ capping and enables translation initiation, the 3’-UTR is indispensable in transcript polyadenylation which in turn influences the stability of mRNA (Chan and Yu, 1998). These untranslated sequences can be manipulated for the optimization of protein expression.
As the protein is synthesized, it undergoes several modifications before final delivery to its cellular destination. These modifications include enzyme involving glycosylation, phosphorylation, methylation, ADP-ribosylation, oxidation, acylation, proteolytic cleavage and non-enzymatic modifications like deamidation, glycation, racemization and spontaneous changes in protein conformation (Gomord & Faye, 2004). Post-translational proteolysis can be effectively minimized by targeting the foreign proteins to sub-cellular compartments such as the endoplasmic reticulum (ER). Proteolysis is more likely to occur in the apoplast and cytosol. ER retrieval signal (e.g. KDEL, HDEL) retains the expressed protein in the ER lumen and has been used to improve foreign protein stability. The ER contains many molecular chaperones facilitating nascent proteins folding or assembly and it is regarded as an ideal compartment for accumulating many classes of foreign proteins (Nuttal et al., 2002).
Other strategies for proteolytic degradation reduction are: co-expression of recombinant protein and protease inhibitors, co-expression of protein co-factors or subunits, knockout mutations in the genes encoding specific proteolytic enzymes.
The recent advent of highly efficient transient expression systems has completely changed the concept and revolutionized plant made pharmaceutical research. Transient transformation implies the expression of foreign DNA which cannot be inherited but is still transcribed within the host cell in a transient manner. Transient gene expression provides a rapid alternative to the time consuming stable transformation methods. This approach uses the plant hosts -
A major breakthrough in viral expression strategies was facilitated by the recent advent of deconstructed virus vectors. Originally reported for the TMV-based magnICON system developed by ICON Genetics GmbH merges advantages of
In conclusion, the two major strategies for expressing proteins in whole plants are transient expression with viral vectors and stable transformation where transgenes are targeted to either the nuclear or chloroplast genome. Stable transformation offers the advantage that protein production is scalable to large field production methods. However, this can be offset by low expression levels and the long time required for creating expressor lines stable across multiple generations. Today’s most promising direction in the referred field is emerging from synthesis of genetically engineered agrobacteria, viruses and plants in one precisely tailored system where synthetic and system biology meet each other.
3. Overview of plant-derived medical recombinant proteins
3.1. Plant derived antibodies
Over the last few decades, medical biotechnology has led to major advances in diagnosis and therapy. At present most diseases can be detected at an early stage, and their treatment is more specific and potent. Biotechnological methods allow to identify the molecular mechanisms of a disease facilitating development of new diagnostic techniques and speeding up development of novel molecularly targeted drugs. One of the therapeutic strategies in the treatment of many diseases is the use of antibodies. Antibodies are a class of topographically homologous multidomain glycoproteins produced by the immune system and they display a remarkably diverse range of binding specificities. Since the first production of monoclonal antibodies by Kohler and Milstein in 1975 they have become an extremely important and valuable tool in medicine (Yarmush et al., 2003).
Constantly increasing demand for new and safe monoclonal antibodies forces development of high-performance production systems. Since the first report on antibody production in
Product | Disease/Pathogen | Plant | Promoter | Expression level | Organ | Reference |
Human anti-rabies monoclonal antibody | Rabies | Tobacco | CaMV 35S promoter with duplicated upstream B domains | 0.07% TSP | Leaves | Ko et al., 2003 |
Human monoclonal antibody | Hepatitis-B virus | Tobacco | CaMV 35S promoter with the omega sequence | 0.2-0.6% TSP | Suspension cell cultures | Yano et al., 2004 |
Full-length monoclonal mouse IgG1 (MGR48) | - | Tobacco | CaMV 35S, TR2' promotor | 30–60 mg of fresh weight | Leaves | Stevens et al., 2000 |
Human-derived, monoclonal antibody | Anthrax | Tobacco | CaMV35S | - | Leaves | Hull et al., 2005 |
Anti-Salmonella enterica single-chain variable fragment (scFv) antibody | Salmonella enterica | Tobacco | EntCUP4, single and double-enhancer versions CaMV 35S | 41.7 ug of scFv/g leaf tissue | Leaves | Makvandi-Nejad et al., 2005 |
Human anti-rabies virus monoclonal antibody | Rabies | Tobacco | CaMV 35S with duplicated upstream B domains (Ca2p), (Pin2p) | 30 ug/g of cell dry weight | Cell suspension culture | Girard et al., 2006 |
BoNT antidotes | Botulinum neurotoxins (BoNTs) | Tobacco | CaMV35S | 20-40 mg/kg | Leaves | Almquist et al., 2006 |
TheraCIM recombinant humanized antibody | Skin cancer | Tobacco | CaMV35S/ Agroinfiltration |
1.2 mg/kg of leaves | Leaves | Rodríguez et al., 2005 |
Human monoclonal antibody 2F5 | Activity against HIV-1 | Tobacco | duplicated CaMV35S | 2.9 ug/g fresh weight | Cell suspension | Sack et al., 2007 |
mAb BR55-2 (IgG2a) | Carcinomas, particularly breast and colorectal cancers | Tobacco | CaMV 35S | 30 mg kg of fresh leaves | Leaves | Brodzik et al., 2006 |
LO-BM2, a therapeutic IgG antibody | Possible tool to prevent graft rejection | Tobacco | En2pPMA4 | 99 ug in the cell extract of a 100-ml culture, 12.81 ug. medium-associated antibody | Leaf and cell suspension culture | De Muynck et al., 2009 |
Monoclonal antibody H10 (mAb H10) | Tumour-associated antigen tenascin-C (TNC) | Tobacco | CaMV 35S with omega translational enhancer sequence from (TMV) | 50–100 mg/kg fresh plant tissue | Leaves | Villani et al., 2009 |
3.2. Plant derived vaccines
Plants can be used to produce inexpensive and highly immunogenic vaccines. It is connected with heterologous expression of antigens. These are further purified to formulate injectable vaccine or are applied as edible vaccines. The latter idea is a very attractive alternative to injection, mostly because of low costs (no need for protein purification) and comfort of administration. However, there are some essential conditions which have to be satisfied. First of all, plants used for oral vaccine production should produce edible parts that can be consumed uncooked (antigens are often heat sensitive). Besides, these parts should be rich in protein because the antigen protein will constitute only a minor portion (0.01-0.4%) of TSP. Seeds seems to be a good choice because of antigen extended stability, even at ambient storage temperatures. As many studies revealed, vaccine antigens present in plant tissues were resistant to digestion in the gastrointestinal tract, on the other hand during this process they were release to elicite both mucosal and systemic immune responses (Sharma and Sood, 2011). Current progress in the matter is summarized in Table 3.
Vaccines | Disease | Plant | Promoter | Expression level | Organ | References | |||||||
Subunit HAC1 and HAI-05 | H1N1, H5N1 influenza | Tobacco | Not reported | HAC1 90 mg/ and HAI-05 50 mg/kg of plant biomass |
Leaves | Shoji et al., 2011 | |||||||
VP1-capsid protein | FMDV ( Foot and Mouth Disease Virus) | Tobacco | psbA | 51% TSP | Leaves (Chloroplasts) | Lentz et al., 2010 | |||||||
TonB protein | Immunization against Helicobacter infections | A. thaliana | CaMV 35S | 0.05% TSP | Entirely plant | Kalbina et al., 2010 | |||||||
Mycobacterial antigens Ag85B | Vaccine against tuberculosis | Tobacco | CaMV 35S | 4 % TSP | Leaves | Floss et al., 2010 | |||||||
Surface protein 4 ⁄ 5 (PyMSP4 ⁄ 5) | Plasmodium | Tobacco | MagnICON® viral vector system | 10% TSP or 1–2 mg⁄g of fresh weight | Leaves | Webster et al. 2009 | |||||||
TetC and PTX S1 antigens | DTP (diphtheria–tetanus–pertussis) | Tobacco Daucus carrota |
CaMV 35S | Not reported | Leaves; Hypocotyls |
Brodzik et al., 2009 | |||||||
HN glycoprotein | Newcastle Disease Virus (NDV) |
Tobacco | P-RbcS | 3µg of HN protein per mg of total leaf protein |
Leaves | Gómeza et al., 2009 | |||||||
HBsAg | HBV (hepatitis B virus) | Lactuca sativa | CaMV 35S | Not reported | Shoots | Marcondes & Hansen, 2008 | |||||||
HPV-16 L1 protein | HPV (Human Papilloma Virus) | Tobacco | psbA promoter | 24 % TSP | Leaves | Fernández-San Millán et al. 2008 | |||||||
16 E7 oncoprotein | HPV | Tomato; Potato |
CaMV 35S | 0.5 % of the cell protein- potato |
Potato protoplast; leaves | Briza et al., 2007 | |||||||
G protein | Rabies virus | Daucus carotta | CaMV 35S | 0.2–1.4% (TSP) | Carrot roots | Royas-Anaya et al., 2009 | |||||||
Capsid protein VP6 | Rotavirus | Potato | P2 | 0.01% | Leaves, tubers | Yu & Landgridge, 2003 |
3.3. Plant derived biopharmaceuticals
Plants can be used to produce inexpensive biopharmaceuticals (Table 4).
Biopharmaceutical | Potential application | Plant | Promoter | Expression level | References |
IL-10 | Inflammatory and autoimmune diseases | Rice seeds | Glutelin B-1 promoter | 2 mg pure IL-10 | Fujiwara et al., 2010 |
Human transfferin | Receptor-mediated endocytosis pathway | Rice seeds | Glutelin 1 G-1 promoter | 1% seed dry weight | Zhang et al., 2010 |
Glutamic acid decarboxylase (GAD65) | Autoimmune T1DM | Tobacco leaves | CaMV 35S | 2.2% total soluble protein | Avesani et al., 2010 |
hGH, somatotropin | Growth hormone-treatment of dwarfism | N. benthamiana | CaMV 35S | 60 mg per kilogram offresh tissue; 7% | Rabindran et. al., 2009; |
Human erythropoietin (EPO) | Anemia, Renal failure | N. tabacum | CaMV 35S | 0.05% of total soluble protein | Conley et al., 2009 |
Human serum albumin (HSA) | Deficiences | Tobacco, potato | Prrn; B33 | 11.1%TSP% (tobacco chloroplasts); 0.2%TSP (potato tuber) | Faran et al., 2002 |
Human lactoferrin (hLF) | Anti-inflammatory and immuno-modulation effects | Potato | Tandem promoter: P2& CaMV 35S | 0.10% TSP | Chong et al., 2000 |
Enkephalins | Painkiller | Cress, A. thaliana | -------------- | 0.10% seed protein | Daniell et al., 2001 |
Staphylokinase | Thrombolytic factor | A. thaliana | CaMV 35S | not reported | Wiktorek-Smagur et al., 2011 |
3.4. Nutraceutical and non-pharmaceutical plant derived proteins
Antimicrobial nutraceutics, such as human lactoferrin and lysozymes, have now been successfully produced in several crops (Stefanova et al., 2008), and are commercially available (Table 5). Cobento Biotechnology (Denmark) has recently received approval for its
Trypsin is a proteolytic enzyme that is used in a variety of commercial applications, including processing of some biopharmaceuticals (Sharma & Sharma, 2009). In 2004, the first plant derived recombinant protein product (bovine sequence trypsin; trade name – trypZean) developed in corn plant (Prodi Gene, USA) was commercialized. Avidin, a glycoprotein found in avian, reptilian and amphibian egg white, is primarily used as a diagnostic reagent. The plant optimized avidin coding sequence was expressed in corn and now it is available on the market. β-glucuronidase, peroxidase, laccase, cellulase, aprotinin were also developed and marketed (Basaran & Rodrigez-Cerezo, 2008).
Spider silk proteins, elastin and collagen, have been expressed in transgenic plants (Scheller et al., 2004). These are promising biomaterials for regenerative medicine.
Product name | Company name | Plant | Commercial name | Source |
Avidin | Prodigene | Corn | Avidin | Obembe at al., 2011 |
β-glucuoronidase | Prodigene | Corn | GUS | Obembe at al., 2011 |
Trypsin | Prodigene | Corn | TrypZean | Obembe at al., 2011 |
Recombinant human lactoferrin | Meristem Therapeutic, Ventria Bioscience |
Corn, Rice | Lacromin | |
Recombinant human lysozyme | Ventria Bioscience | Rice | Lysobac | |
Aprotinin | Prodigene | Corn, Tobacco | AproliZean | Obembe at al., 2011 |
Recombinant lipase | Meristem Therapeutic | Corn | Merispase | .com |
Recombinant human intrinsic factor | Cobento Biotech AS | Arabidopsis | Coban | |
Human growth factors | ORF Genetics | Barley | ISOkineTM | |
Food additive for shrimps | SemBioSys | Safflower | Immuno-spherte |
4. Recombinant protein purification
4.1. Affinity chromatography
Isolation and purification of a biologically active protein from a crude lysate is often difficult and costly. Simple, cheap and more efficient strategies of its purification on the laboratory and industrial scale are thus on great demand. One of the numerous approaches in this field is an affinity tags system easily applicable for recombinant protein purification by affinity chromatography. The term 'affinity chromatography’ was introduced in 1968 by Pedro Cuatrecasas, Meir Wilchek, and Christian B. Anfinsen (1968). Now it is the method of choice (Kabir et al., 2010). Affinity chromatography is based on specific interaction between two molecules in order to isolate the protein of interest from a pool of unwanted proteins and other contaminants. For this purpose a fusion protein is created. A short fragment of DNA can be ligated to the 5 ' or 3' - terminus of the target gene. This peptide or protein coding sequence (so called tag), which is translated in frame with protein of interest exhibits a characteristic property, strong and selective binding to the molecules immobilized on the solid matrices (Fong et al., 2010). Purification process is effective and simple. During passage of the cell extract containing the fusion protein and contaminants through an appropriate column the tagged protein is retained, while all the others migrate freely through the column (Fig. 1).
In the next step, the bound protein is eluted by a change in buffer composition /parameters (i.e. competitors, chelators, pH, ionic strength or temperature). Affinity tags are divided into three main classes according to their properties and the properties of molecules that interact with them: 1) tags, binding to small molecule ligands linked to a solid support (i.e. HIS-tag), 2) protein tags binding to a macromolecular partner immobilized on chromatography support (i.e. CBP-tag), 3) the protein-binding partner attached to the resin in an antibody which recognizes a specific peptide epitope in a recombinant protein (i.e. FLAG-tag) (Lichty et al., 2005, Arnau et al., 2006, Waugh et al., 2005). To date large number of gene fusion tags has been described, the most commonly used ones are presented in Table 6.
Tag | Comments | References |
His-tag | Purification by interaction between immobilized metal ions and chelating amino acids | Valdez-Ortiz et al., 2005, Vaquero et al., 2002 |
FLAG | Purification based on binding the FLAG peptide to antibodies | Brodzik et al., 2009, Zhou and Li., 2005 |
Strep-tag II | Strong specific interaction between Streptag and strep-Tactin (streptavidin derivate) immobilised on resin | Witte et al., 2004 |
4.2. Elastin-like polypeptides in recombinant protein purification
While affinity chromatography is used for purification of a broad spectrum of recombinant proteins it is not free from drawbacks. The main limitations associated with the use of this method are: 1) high cost of chromatography packing materials, 2) volume-limited sample throughput, 3) dilution of the protein product in elution buffer, 4) additional concentration step may cause loss in protein yield (Chow et al., 2008). Taking into account the above, there is a need to introduce new alternative methods for purification of recombinant proteins.
One of the possible solutions is application of non-chromatographic purification tags. Elimination of resins allows us to reduce some of the aforementioned problems.
Elastin-like polypeptides (ELP), artificial polymers containing Val-Pro-Gly-Xaa-Gly pentapeptide repeats, are an example of such tags. Such repeats occur naturally in the hydrophobic domain of human tropoelastin (soluble precursor of elastin) and they play an important role in the process of elastin formation (Mithieux & Weiss 2005, Valiaev et al., 2008). Xaa (so called guest residue) in the ELP sequence can contain any amino acid except for proline (Meyer & Chilkoti, 1999). Occurrence of proline at these positions eliminates distinctive and very useful properties of these polymers (Trabbic-Carlson et al., 2004). Literature classification of ELP is based on the type and number of amino acids present in the guest residue positions (Meyer & Chilkoti 2004).
Elastin-like polypeptides belong to one of the three classes of thermosensitive biopolymers (Mackay and Chilkoti, 2008) whose properties are changed under the influence of moderate temperature differences. Aqueous solutions of ELP exhibit lower critical solution temperature (LCST) which causes that the above phase transition temperature (Tt) ELP pass from soluble to an insoluble form ( Ge et al., 2006 ) in a narrow temperature range (~ 2 C) ( Ge and Filipe, 2006 ). This is a reversible process called coacervation. In solutions with temperature below Tt, free polymer chains remain in a disordered soluble form. The opposite occurs in solutions with temperatures above Tt, when the polymer chains have more ordered structure (called β-helix), stabilized by hydrophobic interactions (Rodriguez-Cabello et al., 2007) that increase association of polymer chains (Serrano et al., 2007). This process is reversible. The fact that ELP –protein fusions are prone to reversible transition is of great importance (Kim et al., 2004). The process of ELP-tagged protein purification involves increasing ionic strength and/or temperature of the cell lysate to induce ELP-fusion protein aggregation (Fig. 2). Next sample centrifugation/filtration separates the ELP fusion protein from contaminants. After resolubilization of an ELP fusion, another centrifugation/filtration removes denatured and aggregated biomolecules. This process called Inverse Transition Cycling (ITC) can be repeated to achieve the required purity of the product ( Floss, Schallau et al., 2010 ).
Purification of proteins using elastin-like polypeptides has several advantages over the traditional chromatographic methods: 1) purification of proteins with ELP tags by ITC appears to be universal for soluble recombinant proteins, 2) chromatography beads are not required, which significantly reduces the costs, 3) final concentration step is not required (Chow et al., 2008).
4.3. Application of ELP to the process of production and purification of recombinant proteins in transgenic plants
Scheller and co-workers (2004) achieved efficient and stable expression of spider’s silk-ELP fusion protein in the ER of transgenic tobacco and potato. Application of ITC allowed them to obtain 80mg pure recombinant protein from 1kg tobacco leaf material. Purified biopolymer was tested as a potential component used for the cultivation of anchorage-dependent CHO-K1 cells and human chondrocytes. The most common coating substances such as collagen, fibronectin and laminin are derived from animal sources, so there is a risk of contamination of cell cultures by viruses or prions which is essentially undesirable in the case of medical applications. What is more, production of this fusion protein in plants is less costly. Lin and associates (2006) obtained active soluble glycoprotein 130 which seems to be potent drug in Crohn’s disease, rheumatoid arthritis and colon cancer therapy. This work a presents creation and expression of mini-gp130-ELP. A fusion protein containing Ig-like domain and cytokine binding module of gp 130 fused to 100 repeats of ELP was expressed in tobacco leaves (ER retention). Inverse transition cycling (ITC) purification resulted in 141
It has been shown for spider silk proteins (Scheller et al., 2004), murine interleukin-4, human interleukin-10 (Patel et al., 2007) and anti-HIV type 1 antibodies (Floss et al., 2008, Floss et al., 2009) that the ELP fusion significantly enhances accumulation of recombinant proteins produced in plants. So far the mechanism of that phenomenon is not known.
5. Status of plant-derived biopharmaceuticals in clinical development
At present some non-pharmaceutical products from plants are on the market (Basaran and Rodriguez-Cerezo, 2008). Although no plant made pharmaceutical (PMP) has been commercialized as a human drug, several PMPs are at the late stage of development and some have already received regulatory approval, including a vaccine and several nutraceuticals (Table,7, 8, 9).
Antibodies | Target | Plant | Clinical trial status | Company | Source |
DoxoRx | Side-effects of cancer therapy | Tobacco | Phase I | Planet Biotechnology | biotechnology.com |
RhinoRX | Common cold | Tobacco | Phase I | Planet Biotechnology | biotechnology.com |
IgG (ICAM1) | Common cold | Tobacco | Phase I | Planet Biotechnology | biotechnology.com |
CaroRX | Dental caries | Tobacco | EU approved as medical advice | Planet Biotechnology, | biotechnology.com |
Antigen or vaccine | Disease | Plant | Clinical trial status | Company | Source |
Hepatitis B antigen | Hepatitis B | Lettuce | Phase I | Thomas Jefferson University | Streatfield, 2006 |
Hepatitis B antigen | Hepatitis B | Potato | Phase II | Arizona State University | Streatfield, 2006 |
Fusion proteins | Rabies | Spinach | Phase I | Thomas Jefferson University | http://www.labome.org |
Heat labile toxin B subunit of E.coli | Diarrhea | Potato | Phase I | ProdiGene | Tacket, 2005 |
Capsid protein Norwalk virus | Diarrhea | Potato | Phase I | Arizona State University | Khalsa et al., 2004 |
Vibrio cholerae | Cholera | Potato | Phase I | Arizona State University | Tacket, 2005 |
HN protein of Newcastle disease virus | Newcastle disease (Poultry) | Tobacco | USDA Approved | Dow Agro Sciences | http://www.dowagro.com |
Viral vaccine mixture | Diseases of horses, dogs |
Tobacco | Phase I | Dow Agro Sciences | http://www.dowagro.com |
Poultry vaccine | Coccidiosis infection | Canola | Phase II | Guardian Bioscence | Basaran & Rodrigez-Cerezo, 2008 |
Gastroenteritis virus (TGFV) capsid protein | Piglet gastroenteritis | Maize | Phase I | ProdiGene | Basaran & Rodrigez-Cerezo, 2008 |
H5N1 vaccine candidate | H5N1 pandemic influenza | Tobacco | Phase I | Medicago | http://www.medicago.com |
Therapeutic humans protein | Disease | Plant | Clinical trial status | Company | Source |
α-Galactosidase | Fabry disease | Tobacco | Phase I | Planet Biotechnology | biotechnology.com |
Lactoferon | Hepatitis C | Duckweed | Phase II | Biolex | http://www.biolex.com |
Fibrinolytic drug | Blood clot | Duckweed | Phase I | Biolex | http://www.biolex.com |
Human glucocerebrosidase | Gaucher’s disease | Carrot | Waiting USDA’s approval | Prostalix Biotherapeutic | http.//www.prostalix.com |
Insulin | Diabetes | Safflower | Phase III | SemBioSys | http.//www.sembiosysys.com |
Apolipoprotein | Cardio vascular |
Safflower | Phase I | SemBioSys | http.//www.sembiosysys.com |
In 2006 the world’s first plant made vaccine candidate for Newcastle disease in chickens, produced in a suspension cultured tobacco cell line by Dow Agro Science, was registered and approved by the US Department of Agriculture (USDA) – the final authority for veterinary vaccines. In addition, two plant made pharmaceuticals are moving through Phase II and Phase III human clinical trials. Biolex’s product candidate, Locteron®, is in Phase IIb clinical testing for the treatment of chronic hepatitis CA. This company uses two genera,
Medicago Inc. of Canada was invited to the sixth WHO meeting about evaluation of pandemic influenza prototype vaccines in clinical trials. One of the purposes of this meeting was to make recommendations on research activities that will contribute to the development of effective pandemic vaccines. Medicago has recently reported positive results from a Phase I human clinical trial with its H5N1 avian influenza vaccine candidate (a VLP based vaccine produced with a transient expression system). The vaccine was found to be safe, well tolerated and it also induced a solid immune response. Based on these results, Medicago will process with Phase II clinical trial with the first plant made influenza vaccine (Franconi et al., 2010). These examples will pave the way to easy public acceptance of transgenic plants as new production platforms for human therapeuticals.
6. Concluding remarks
Biopharming is still a relatively new field in plant science but in the coming years it may become the premier expression system for a wide variety of new biopharmaceuticals. The use of plants as factories for the synthesis of therapeutic protein molecules will undoubtedly develop. Since the first development of a genetically modified plant in 1984, numerous comprehensive review articles have been published demonstrating the tremendous potential of plants for pharmaceutical production. As it has been clearly shown plants are no longer considered only in terms of diet or beauty. The proteins targeted for biopharmaceutical technology form three broad categories: antibodies, vaccines, and other therapeutics. Plant bioreactors represent an attractive alternative for their synthesis requiring the lowest capital investment of all tested production systems. The events of heterologous proteins in planta production were rapidly followed with development/improvement of significant technologies (e.g. DNA delivery systems, selection methods). At present a number of promoters with tissue-specific activity or sub-cellular targeting sites that offer protein stability are known and many are still under intense study. Obviously, the construction of a transgenic plant synthesizing a functional therapeutic is a multidisciplinary process and the society of biotechnologists takes a keen interest in its success. However, over the past years various plant expression platforms have been tested and it is evident that further development and improvement are needed for more effective molecular farming. Apart from continuously increasing transgene yields efforts will need to ensure that plant-derived biopharmaceuticals would meet the same safety and efficacy standards as products of non-plant origin. There is no doubt that sooner or later the scientific limitations of molecular farming will be overcome, especially when numerous therapeutics and plant platforms are developed by many laboratories and companies. Thus, this is the regulatory requirements and public acceptance which are the greatest challenge of modern plant biotechnology. Of course, molecular farming raises less objection than technologies using genetically modified animals, but still the existing or proposed regulations remain based on public fears rather than on scientific facts.
In conclusion, “the molecular farming industry” means a natural advance in drug production technology. The dynamics of optimization and improvement of plant expression platforms illustrates its potential and tremendous scientific background. The possible success in this field will have to face the question of public acceptance. Thus, the scientists should send the clear massage to the public opinion that molecular farming is a strictly controlled technology that has strong benefits. And that probably will be more difficult than the construction of functional bioreactor itself.
References
- 1.
Almquist K. C. MD Mc Lean Niu Y. Byrne G. Olea-Popelka F. C. Murrant C. Barclay J. Hall J. C. 2006 Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco . ,24 12 (December, 2006),2079 2086 ,0026-4410 X - 2.
Arnau J. Lauritzen C. Petersen G. E. Pedersen J. 2006 Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins . ,48 1 (July 2006)1 13 ,1046-5928 - 3.
Avesani A. Vitale A. Pedrazzini E. de Virgilio M. Pompa A. Barbante A. Gecchele E. Dominici P. Morandini F. Brozzetti A. Falorni A. Mario Pezzotti. M. 2010 Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant. ,8 8 (September 2010),862 872 ,1467-7644 - 4.
Basaran P. Rodriguez-Cerezo E. 2008 Plant molecular farming: opportunities and challenges. ,28 3 (March 2008),153 172 ,1549-7801 - 5.
Boehm R. 2007 Bioproduction of Therapeutic Proteins in the 21st Century and the Role of Plants and Plant Cells as Production Platforms . ,1102 (April 2007),121 134 ,1749-6632 - 6.
Briza J. Pavingerowa D. Vlasak J. Ludvikova V. Niedermeierova H. 2007 Production of human papillomavirus type 16 E7 oncoprotein fused with β-glucuronidase in transgenic tomato and potato plants.,51 2 (June 2007),268 276 ,0006-3134 - 7.
Brodzik R. Glogowska M. Bandurska K. Okulicz M. Deka D. Ko K. van der Linden J. Leusen J. H. Pogrebnyak N. Golovkin M. Steplewski Z. Koprowski H. 2006 Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells . ,103 23 (May 2006),8804 8809 ,0027-8424 - 8.
Brodzik R. Spitsin S. Pogrebnyak N. Bandurska K. Portocarrero C. Andryszak K. Koprowski H. Golovkin M. 2009 Generation of plant-derived recombinant DTP subunit vaccine . ,27 28 (June 2009),3730 3734 ,0026-4410 X - 9.
Cardi T. Lenzi P. Maliga P. 2010 Chloroplast as expression platforms for plant produced vaccines.9 8 (October 2009),893 911 ,1476-0584 - 10.
Chan M. T. Yu S. M. 1998 The 3’ untranslated region of a rice alpha amylase gene function as a sugar dependent mRNA stability determinant. ,95 11 (May 1988),6543 6547 ,0027-8424 - 11.
Chen Z. L. MA Schuler RN Beachy 1986 Functional analysis of regulatory elements in a plant embryo specific gene . ,83 22 (November 1986),8560 9564 ,0027-8424 - 12.
Chong D. K. X. Langridge W. H. R. 2000 Production of full- length bioactive antimicrobial human lactoferrin in potato plants.9 1 (January 2000),71 78 ,0962-8819 - 13.
Chow D. Nunalee M. L. Lim D. W. Simnick A. J. Chilkoti A. 2008 Peptide-based Biopolymers in Biomedicine and Biotechnology .62 4 (January 2008)125 155 ,0000-0927 -796X - 14.
Conley A. J. Mohib K. Jevnikar A. M. Brandle J. E. 2009 Plant recombinant erythropoietin attenuates inflammatory kidney cell injury . ,7 2 (November 2009),183 199 ,1467-7644 - 15.
Cowen N. M. Smith K. A. Armstrong K. 2007 Use of regulatory sequences in transgenic plants. Unitated States Patent,7179902 - 16.
Cox K. M. Sterling J. D. Regan J. T. Gasdaska J. R. Frantz K. K. Peele C. G. Black A. Passmore D. Moldovan-Loomis C. Srinivasan M. Cuison S. Cardelli P. M. Dickey L. F. 2006 Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. ,24 11 November 2006)1591 1597 ,1087-0156 - 17.
Cramer C. L. Weissenborn D. L. Oishi K. K. Grabau E. A. Benett S. Ponce E. Grabowski G. A. Radin D. N. 1996 Bioproduction of human enzymes in transgenic tobacco. 729 1 (May 1996),62 71 ,0077-8923 - 18.
Cuatrecasas P. Wilchek M. Anfinsen C. B. 1968 Selective enzyme purification by affinity chromatography . ,61 2 (October 1968)636 643 , ISSN-0027-8424 - 19.
Dai Z. BS Hooker Anderson D. B. Thomas S. R. 2000 Improved plant based production of E1 endoglucanase using potato: expression.6 3 (June 2000),277 285 ,1380-3743 - 20.
Daniell H. Streatfield S. J. Wycoff K. 2001 Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. ,6 5 May 2001),219 226 ,1360-1385 - 21.
Decker E. L. Reski R. 2008 Current achievements in the production of complex biopharmaceuticals with moss bioreactors .31 1 (January 2008),3 9 ,0016-1576 - 22.
Demain A. L. Vaishnav P. 2009 Production of recombinant proteins by microbes and higher organism. ,27 3 (June 2009),297 306 ,0734-9750 - 23.
De Muynck B. Navarre C. Nizet Y. Stadlmann J. Boutry M. 2009 Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells . ,18 3 (January 2009),467 482 ,0962-8819 - 24.
Fernández-San Millán. A. Ortigosa S. M. Hervás-Stubbs S. Corral-Martínez P. Seguí-Simarro J. M. Gaétan J. Coursaget P. Veramendi J. 2008 Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic . ,6 6 (April 2008),427 441 ,1467-7644 - 25.
Fischer R. Emans N. 2000 Molecular farming of pharmaceutical proteins . ,9 4-5 . (August 2000),279 299 ,1573-9368 - 26.
Farran I. Sánchez-Serrano J. J. Medina J. F. Prieto J. Mingo-Casel A. M. 2002 Targeted expression of human serum albumin to potato tubers. ,11 4 (August 2002),337 346 ,0962-8819 - 27.
Floss D. M. Mockey M. Zanello G. Brosson D. Diogon M. Frutos R. Bruel T. Rodrigues V. Garzon E. Chevaleyre C. Berri M. Salmon H. Conrad U. Dedieu L. 2010 Expression and immunogenicity of the mycobacterial Ag85B/ESAT-6 antigens produced in transgenic plants by elastin-like peptide fusion strategy. 2010:274346. Epub 2010 Apr 13,1110-7243 1110 7243 - 28.
Floss D. M. Sack M. Arcalis E. Stadlmann J. Quendler H. Rademacher T. Stoger E. Scheller J. Fischer R. Conrad U. 2009 Influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody . ,7 9 (December 2009),899 913 ,1467-7644 - 29.
Floss D. M. Sack M. Stadlmann J. Rademacher T. Scheller J. Stöger E. Fischer R. Conrad U. 2008 Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants . ,6 4 (May 2008),379 391 ,1467-7644 - 30.
Floss D. M. Schallau K. Rose-John S. Conrad U. Scheller J. 2010 Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application . ,28 1 (January 2010),37 45 ,0167-7799 - 31.
Fogher C. 2000 A synthetic polynucleotide coding for human lactoferrin, vectors, cells and transgenic plants containing it. Gene bank accAX006477 Patent: W00004146. - 32.
BA Fong Wu W. Y. Wood D. W. 2010 The potential role of self-cleaving purification tags in commercial-scale processes . ,28 5 (May 2010)272 279 ,0167-7799 - 33.
Franconi R. Demurtas O. C. Massa S. 2010 Plant derived vaccines and other therapeutics produced in contained systems. ,9 8 (October 2010),877 892 ,1476-0584 - 34.
Fujiwara Y. Aiki Y. Yang L. Takaiwa F. Kosaka A. Tsuji N. M. Shiraki K. Sekikawa K. 2010 Extraction and purification of human interleukin-10 from transgenic rice seeds . ,72 1 (February 2010),125 130 ,1046-5928 - 35.
Ge X. Filipe C. D. 2006 Simultaneous phase transition of ELP tagged molecules and free ELP: an efficient and reversible capture system. ,7 9 (Septrember 2006),2475 2478 ,1525-7797 - 36.
Ge X. Trabbic-Carlson K. Chilkoti A. Filipe C. D. M. 2006 Purification of an elastin-like fusion protein by microfiltration. ,95 3 (October 2006),424 432 46-851,0006-3592 - 37.
Girard L. S. Fabis M. J. Bastin M. Courtois D. Pétiard V. Koprowski H. 2006 Expression of a human anti-rabies virus monoclonal antibody in tobacco cell culture . ,345 2 (January 2006)602 607 ,0000-6291 X - 38.
Gleba Y. Klimyuk V. Marillonnet S. 2005 Magnifection- a new platform for expressing recombinant vaccines in plants. ,23 17-18 , (March 2005),2042 2048 ,0026-4410 X. - 39.
Gómeza E. Zotha S. Ch Asurmendia S. Vázquez Roverea. C. Berinstein A. 2009 Expression of Hemagglutinin-Neuraminidase glycoprotein of Newcastle Disease Virus in agroinfiltrated plants. Journal of Biotechnology,144 4 (September 2009),337 340 ,0168-1656 - 40.
Gomord V. Faye L. 2004 Posttranslational modification of therapeutic proteins in plants. ,7 2 (April 2004),171 181 ,1369-5266 - 41.
Halweg C. Thompson W. F. Spiker S. 2005 The rb7 matrix attachment region increase the likehood and magnitude of transgene expression in tobacco cells: a flow cytometric study. ,17 2 (February 2005),418 429 ,1040-4651 - 42.
Hearn M. T. Acosta D. 2001 Applications of novel affinity cassette methods: use of peptide fusion handles for the purification of recombinant proteins . ,14 6 (December 2001),323 369 ,0000-0952 -3499 - 43.
Hellwig S. Drossard J. Twyman R. M. Fischer R. 2004 Plant cell cultures for the production of recombinant proteins. ,22 11 (November 2004),1415 1422 ,1087-0156 - 44.
Herman S. R. Harding R. M. Dale J. L. 2001 The banana promoter drives near constitutive transgene expression in vegetative tissue of banana (Musa spp.). Plant Cell Report,20 6 (July 2001),525 530 ,0721-7714 - 45.
Hiatt A. Cafferkey R. Bowdish K. 1989 Production of antibodies in transgenic plants. ,342 6245 (July-August 1989),76 78 ,0028-0836 - 46.
Huang Z. Santi L. Le Pore K. Kilbourne J. Arntzen C. J. Mason H. S. 2006 Rapid, high level production of hepatitis B core antigen in plant leaf and its immunogenicity in mice. ,24 14 (December 2006),2506 2513 ,0026-4410 X. - 47.
Hull A. K. Criscuolo C. J. Mett V. Groen H. Steeman W. Westra H. Chapman G. Legutki B. Baillie L. Yusibov V. 2005 Human-derived, plant-produced monoclonal antibody for the treatment of anthrax. ,23 17-18 , (March 2005),2082 2086 ,0026-4410 X - 48.
Jeferson R. Goldsbrough A. Bevan M. 1990 Transcriptional regulation of a gene in potato. Plant Molecular Biology,14 6 (February 1990),995 1006 ,0735-9640 - 49.
Jiang X. L. He Z. M. Peng Z. Q. Qi Y. Chen Q. You S. Y. 2007 Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice . ,16 2 (April 2007),169 175 ,1573-9368 - 50.
Joensuu J. J. Brown K. D. Conley A. J. Clavijo A. Menassa R. Brandle J. E. 2009 Expression and purification of an anti-Foot-and-mouth disease virus single chain variable antibody fragment in tobacco plants . ,18 5 (October 2009),685 696 ,0962-8819 - 51.
Kabir M. E. Krishnaswamy S. Miyamoto M. Furuichi Y. Komiyama T. 2010 Purification and functional characterization of a Camelid-like single-domain antimycotic antibody by engineering in affinity tag . ,72 1 (July 2010),59 65 ,1046-5928 - 52.
Kaiser J. 2008 Is the drought over for pharming? ,320 5875 (April 2008),473 475 ,1095-9203 - 53.
Kalbina I. Engstrand L. Andersson S. Strid A. (2010 Expression of Helicobacter pylori TonB Protein in Transgenic Arabidopsis thaliana: Toward Production of Vaccine Antigens in Plants. ,15 15 5 (October 2010),430 437 ,1523-5378 - 54.
Khalsa G. Mason H. S. Arntzen C. J. 2004 Plant derived vaccines: progress and constrains, In: , R. Fischer, S. Schillberg, (Ed),135 158 , Wiley-VCH,978-3-52760-363-3 Weinheim, Germany. - 55.
Kim J. Y. Mulchandani A. Chen W. 2004 Temperature-triggered purification of antibodies. ,90 3 (May 2004),373 379 ,0006-3592 - 56.
Ko K. Tekoah Y. Rudd P. M. Harvey D. J. Dwek R. A. Spitsin S. CA Hanlon Rupprecht C. Dietzschold B. Golovkin M. Koprowski H. 2003 Function and glycosylation of plant-derived antiviral monoclonal antibody . ,100 13 (June 2003),8013 8018 ,0027-8424 - 57.
Komarova T. V. Baschieri S. Donini M. Marusic C. Benvenuto E. Dorokhov Y. L. 2010 Transient expression system for plant derived biopharmaceuticals. ,9 8 (October 2009),859 876 ,1476-0584 - 58.
Lentz E. M. Segretin M. E. Mauro M. Wirth S. A. Mozgovoj M. V. Wigdorovitz A. Bravo-Almonacid F. F. 2010 High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants . ,231 2 (November 2010),387 395 ,0032-0935 - 59.
Lichty J. J. Malecki J. L. Agnew H. D. Michelson-Horowitz D. J. Tan S. 2005 Comparison of affnity tags for protein purifcation. ,41 1 (May 2005),98 105 ,1046-5928 - 60.
Lin M. Rose-John S. Grötzinger J. Conrad U. Scheller J. 2006 Functional expression of a biologically active fragment of soluble gp130 as an ELP-fusion protein in transgenic plants: purification via inverse transition cycling. ,398 3 (September 2006),577 583 ,0264-6021 - 61.
Ling H. Y. Pelosi A. Walmsley A. M. 2010 Current status of plant made vaccines for veterinary purposes . ,9 8 (October 2009),971 982 ,1476-0584 - 62.
Mackay J. A. Chilkoti A. 2008 Temperature sensitive peptides: engineering hyperthermia-directed therapeutics . : the Official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group,24 6 (September 2008),846 851 ,0265-6736 - 63.
Makvandi-Nejad S. Mc Lean M. D. Hirama T. Almquist K. C. Mackenzie C. R. Hall J. C. 2005 Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B . ,14 5 (October 2005),785 792 ,0962-8819 - 64.
Marcondes J. Hansen E. (2008 12 12 6 (December 2008),469 471 ,1413-8670 - 65.
Medrano G. MJ Reidy Liu J. Ayala J. Dolan M. C. Cramer C. L. 2009 Rapid system for evaluating bioproduction capa city complex pharmaceutical proteins in plants. ,483 (January 2008),51 67 ,1064-3745 - 66.
Menassa R. Zhu H. Karatzs C. N. Lazaris A. Richman A. Brandle J. 2004 Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production . ,2 5 (September 2004),431 438 ,1229-2818 - 67.
Meyer D. E. Chilkoti A. 1999 Purification of recombinant proteins by fusion with thermally-responsive polypeptides. .17 11 (January 1999)1112 1115 ,1087-0156 - 68.
Meyer D. E. Chilkoti A. 2004 Quantification of the effect of chain length and concentration on the thermal behavior of elastin-like polypeptides. ,5 3 (May- June 2004),846 851 ,1525-7797 - 69.
Mithieux S. M. Weiss A. S. 2005 Elastin. ,70 437 461 ,0065-3233 - 70.
Muto M. Ryan H. E. Mayfield S. P. 2009 2009).Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast. ,9 (March 2009),26 doi:10.1186/1472-6750-9-26, 1472-6750 - 71.
Ohana R. F. Encell L. P. Zhao K. Simpson D. Slater M. R. Urh M. Wood K. V. 2009 HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification . ,68 1 (November 2009),110 120 ,1046-5928 - 72.
Osborn T. C. Burrow M. Bliss F. A. 1988 Purification and characterization of arcelin seed protein from common bean. ,86 2 (February 1988),399 405 ,234547-6478 - 73.
Patel J. Zhu H. Menassa R. Gyenis L. Richman A. Brandle J. 2007 Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves . ,16 2 (April 2007)239 249 ,0962-8819 - 74.
Rabindran S. Roy N. Fedorkin G. Skarjinskaia M. 2009 Plant-Produced Human Growth Hormone Shows Biological Activity in a Rat Model . ,25 2 (March 2009),530 534 ,1520-6033 - 75.
Rival S. Wisniewski J. P. Langlais A. Kaplan H. Freyssinet G. Vancanneyt G. Vunsh R. Perl A. Edelman M. 2008 Spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin.17 4 (August 2008),503 513 ,0962-8819 - 76.
Rodríguez M. Ramírez N. I. Ayala M. Freyre F. Pérez L. Triguero A. Mateo C. Selman-Housein G. Gavilondo J. V. Pujol M. 2005 Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor. ,89 2 (January 2005),188 194 ,0006-3592 - 77.
Rodriguez-Cabello J. C. Prieto S. Reguera J. Arias F. J. Ribeiro A. 2007 Biofunctional design of elastin-like polymers for advanced aplications in nanobiotechnology. J18 3 (March 2007),269 286 ,0920-5063 - 78.
Rojas-Anaya E. Loza-Rubio E. Olivera-Flores M. T. Gomez-Lim M. 2009 Expression of rabies virus G protein in carrots (Daucus carota)18 6 (December 2009),911 919 ,0962-8819 - 79.
Sack M. Paetz A. Kunert R. Bomble M. Hesse F. Stiegler G. Fischer R. Katinger H. Stoeger E. Rademacher T. 2007 Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures. he ,21 8 (June 2007)1655 1664 ,0892-6638 - 80.
Scheller J. Hengger D. Viviani A. Conrad U. 2004 Purification of spider silk elastin from transgenic plants and application for human chondrocyte proliferation . ,13 1 (February 2004)51 57 ,1573-9368 - 81.
Serrano V. Liu W. Franzen S. 2007 An infrared spectroscopic study of the conformational transition of elastin-like polypeptides. ,93 7 (October 2007),2429 2435 ,0006-3495 - 82.
Sharma A. K. Sharma M. K. 2009 Plants as bioreactors: Recent developments and emerging opportunities . ,27 6 (June 2009),811 832 ,1476-0584 - 83.
Sharma M. Sood B. 2011 A banana or syringe: journey to edible vaccines. ,27 3 (June 2011),471 477 ,1573-0972 - 84.
Shoji Y. Chichester J. A. Jones M. Manceva S. D. Damon E. Mett V. Musiychuk K. B. Farrance H. Ch Shamloul M. Kushnir N. Sharma S. Vidadi Y. V. 2011 Plant-based rapid production of recombinant subunit hemagglutinin vaccines targeting H1N1 and H5N1 influenza.7 Supplement, (January/February 2011),41 50 ,1554-8600 - 85.
Smith M. L. Mason H. S. Shuler M. L. 2002 Hepatis B surface antigen (HBsAg) expression in plant cell culture: kinetics of antigen accumulation in batch culture and its intracellular form.80 7 (December 2002),812 822 ,0006-3592 - 86.
Steen J. Uhlén M. Hober S. Ottosson J. 2006 High-throughput protein purification using an automated set-up for high-yield affinity chromatography . ,46 2 173 178 ,1046-5928 - 87.
Stefanov I. Illubaev S. Feher A. Margoczi K. Dudits D. 1991 Promoter and genotype dependent transient expression of a reporter gene in plant protoplasts. ,42 4 (April 1991),323 330 ,0236-5383 - 88.
Stefanova G. Vlahlova M. Atanassov A. 2008 Production of recombinant human lactoferrin from transgenic plants .52 3 (May 2008),423 428 ,1573-8264 - 89.
Stevens L. H. Stoopen G. M. Elbers I. J. Molthoff J. W. Bakker H. A. Lommen A. Bosch D. Jordi W. 2000 Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. ,124 1 ( May 2000),173 182 ,0032-0889 - 90.
Stoger E. Sack M. Perrin Y. Vaquero C. Torres E. Twyman R. M. Christou P. Fischer R. 2002 Practical considerations for pharmaceutical antibody production in different crop systems.9 3 (September 2002),149 158 ,1380-3743 - 91.
Streatfield S. J. 2006 Mucosal immunization using recombinant plant based oral vaccines . ,38 2 (February 2006),150 157 ,1046-2023 - 92.
Tacket C. O. 2005 Plant derived vaccines against diarrheal diseases. ,23 15 (March 2005),1866 1869 ,0026-4410 X. - 93.
Thanavala Y. Huang Z. Mason H. S. 2006 Plant derived vaccines: a look back at the highlights and a view to the challenges on the road ahead. ,5 2 (February 2006),249 260 ,1476-0584 - 94.
Trabbic-Carlson K. Meyer D. E. Liu L. Piervincenzi R. Nath N. La Bean T. Chilkoti A. 2004 Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?17 1 (August 2004),57 66 ,1741-0126 - 95.
Valdez-Ortiz A. Rascón-Cruz Q. Medina-Godoy S. Sinagawa-García S. R. Valverde-González M. E. Paredes-López O. 2005 One-step purification and structural characterization of a recombinant His-tag 11S globulin expressed in transgenic tobacco. ,115 4 (February 2005),413 423 ,0168-1656 - 96.
Valiaev A. Lim D. W. Schmidler S. Clark R. L. Chilkoti A. Zauscher S. 2008 Hydration and conformational mechanics of single, end-tethered elastin-like polypeptides. J,130 33 (July 2008),10939 10946 ,0002-7863 - 97.
Vaquero C. Sack M. Schuster F. Finnern R. Drossard J. Schumann D. Reimann A. Fischer R. 2002 A carcinoembryonic antigen-specific diabody produced in tobacco. ,16 3 (January 2002),408 410 ,0000-0892 -6638 - 98.
Villani M. E. Morgun B. Brunetti P. Marusic C. Lombardi R. Pisoni I. Bacci C. Desiderio A. Benvenuto E. Donini M. 2009 Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies . ,7 1 (January 2009),59 72 ,0000-1467 -7644 - 99.
DS Waugh 2005 Making the most of affinity tags. ,23 6 (June 2005),316 320 ,0167-7799 - 100.
Webster D. E. Wang L. Mulcair M. Ma Ch Santi L. Mason H. S. Wesselingh S. L. Ross L. Coppel R. L. 2009 Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus-based expression system .7 9 (December 2009),846 855 ,1467-7644 - 101.
Wiktorek-Smagur A. Hnatuszko-Konka K. Gerszberg A. Łuchniak P. Kowalczyk T. Kononowicz A. K. 2011 Expression of a staphylokinase, a thrombolytic agent in . World Journal Microbiology and Biotechnology,27 6 (June 2011),1341 1347 ,0959-3993 - 102.
CP Witte Noël L. D. Gielbert J. Parker J. E. Romeis T. 2004 Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope . ,55 1 (May 2004),135 147 ,0000-0167 -4412 - 103.
Wu Cy. Suzuki A. Washida H. Takaiwa F. 1988 The GCN4 motif in a rice glutelin gene is essential for endosperm specific gene expression and is activated by Opaquae-2 in transgenic rice plants.14 6 (June 1988),673 683 ,0136-5313 X. - 104.
Xie Y. Liu Y. Meng M. Chen L. Zhu Z. 2003 Isolation and identification of a super strong plant promoter from cotton leaf curl Multan virus .53 1-2 , (July 2003),1 14 ,0735-9640 - 105.
Xu J. Ge X. Dolan M. C. 2011 Towards high-yield production of pharmaceuticals proteins with plant cell suspension culture. ,29 3 (January 2011),278 299 ,0734-9750 - 106.
Yano A. Maeda F. Takekoshi M. 2004 Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen.73 2 (June 2004),208 215 ,0146-6615 - 107.
Yap Y. K. Smith D. R. 2010 Strategies for the plant based expression of dengue subunit vaccines . ,57 2 (December 2010),47 53 ,0885-4513 - 108.
Yarmush M. L. Toner M. Plonsey R. Bronzino J. D. 2003 Monoclonal Antibodies and Their Engineered Fragments In:1 17 , CRC Press; 1 edition (March 26, 2003),0-84931-811-4 - 109.
Yu J. Langridge W. 2003 Expression of rotavirus capsid protein VP6 in transgenic potato and its oral immunogenicity in mice. ,12 2 (April 2003),163 169 ,1573-9368 - 110.
Yusibov V. Rabindran S. 2008 Recent progress in the development of plant derived vaccines . ,7 8 (October 2008),1173 1183 ,1476-0584 - 111.
Zhang D. Nandi S. Bryan P. Pettit S. Nguyen D. MA Santos Huang N. 2010 Expression, purification, and characterization of recombinant human transferrin from rice ( L.). Protein Expression and Purification,74 1 (November 2010),69 70 ,1046-5928 - 112.
Zhou A. Li J. 2005 Arabidopsis BRS1 is a secreted and active serine carboxypeptidase. ,280 42 (October 2005),35554 355561 ,0021-9258 - 113.
Zuo J. Chua N. H. 2000 Chemical-inducible systems for regulated expression of plant genes. ,11 2 (April 2000),146 151 ,0958-1669