Biotechnology Advances 28 (2010) 427–435

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Production of heterologous proteins in plants: Strategies for optimal expression
Priti N Desai, Neeta Shrivastava, Harish Padh ⁎
B. V. Patel Pharmaceutical Education and Research Development Centre, Ahmedabad, India

a r t i c l e

i n f o

Article history:
Received 3 September 2009
Received in revised form 1 January 2010
Accepted 25 January 2010
Available online 10 February 2010
Keywords:
Heterologous proteins
Plant expression systems
Protein production
Protein accumulation
Therapeutic proteins

a b s t r a c t
Plants are a promising expression system for the production of heterologous proteins, especially therapeutic
proteins. Currently the majority of therapeutic proteins are produced in mammalian cell lines or bacteria. In
a few cases insects, yeast and fungi have been developed for production of human proteins. However, these
expression systems have limitations in terms of suitability, cost, scalability, purification and posttranslational modifications. Therefore, alternative expression systems are being developed in transgenic
animals and transgenic plants. Transgenic plants could provide an attractive alternative in terms of low
production cost and lower capital investment in infrastructure, and with appropriate post-translational
modifications. The potential of plants as an expression host has not been capitalized, primarily due to lower
level of expression of transgenes in plants. The present review will evaluate the rate limiting steps of plant
expression systems and suggest strategies to optimize protein expression at each of the steps: gene
integration, transcription, translation and protein accumulation.
© 2010 Elsevier Inc. All rights reserved.

Contents
1.

Introduction . . . . . . . . . . . . . . . . . . . . .
1.1.
Heterologous proteins . . . . . . . . . . . . .
1.2.
Expression systems . . . . . . . . . . . . . .
1.3.
Commercial demand of therapeutic proteins . .
2.
Plants as an expression system . . . . . . . . . . . .
2.1.
Existing plant-based technologies . . . . . . .
2.2.
Advantages of plant expression systems . . . .
2.3.
Limitations of plant expression systems . . . .
3.
Factors influencing the heterologous protein production
3.1.
Choice of expression vectors . . . . . . . . . .
3.2.
Integration of foreign gene . . . . . . . . . .
3.3.
Transcription . . . . . . . . . . . . . . . . .
3.3.1.
Transcription initiation . . . . . . . .
3.3.2.
RNA processing . . . . . . . . . . . .
3.3.3.
RNA stability . . . . . . . . . . . . .
3.4.
Translation . . . . . . . . . . . . . . . . . .
3.4.1.
Translation initiation . . . . . . . . .
3.4.2.
Elongation and termination . . . . . .
3.5.
Final yield or protein accumulation . . . . . . .
3.5.1.
Proteases . . . . . . . . . . . . . . .
3.6.
Gene silencing . . . . . . . . . . . . . . . .
4.
Summary and concluding remarks . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. B. V. Patel Pharmaceutical Education and Research Development Centre, Thaltej-Gandhinagar Highway, Thaltej, Ahmedabad 380054, India. Tel.: + 91 79
2743 9375; fax: + 91 79 2745 0449.
E-mail address: perd@perdcentre.com (H. Padh).
0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2010.01.005

2007. 2003. Rai & Padh. (h) Donald & Jarvis. proteins used as reagents for research and study purposes and those proteins with various industrial applications. Wildt and Gernfross. . (e) Walsh. Heterologous proteins are divided into three major groups: therapeutic proteins or those used for clinical diagnosis. 1981. phosphorylation and proper folding. Fig. Gradually various changes were introduced in the E. r-Protein (recombinant protein). Demonstrates advances in various expression systems with reported milestones. which also involves post-translational modifications of protein. 2004. was successfully expressed (Itakure et al... ATryn (recombinant human anti thrombin produced in goat by GTC Biotherapeutics). References: (a) Itakure et al. / Biotechnology Advances 28 (2010) 427–435 1. 2007. a suitable vector and a biological system (expression system) which can transcribe and translate the transgene into a desired protein. Expression systems Molecular biology tools have helped us cross the species barrier and produce proteins of our interest in species of our choice. 1983. 2005). (f) Wildt & Gernfross. 2005. (ii) have good productivity. 2004. 1981. (n) Moreira.. (g) Smith et al... (o) Wurm. The essential tools for production of heterologous proteins are a gene or cDNA encoding desired protein... Strasser et al. Heterologous proteins In vivo production of protein is a very complex process. tPA (human tissue plasminogen activator). 2007. coli expression system (Pillai and Panchagnula. (l) Simons et al. Hence. 1986. Insulin was the first FDA approved therapeutic protein produced from E. 1983. Introduction 1. coli and mammalian cell expression systems are main bioreactors for the production of therapeutic proteins. 2004). required for its stability and biological activity: like glycosylation. and (v) afford easy downstream processing. 1988) were gradually involved. This single capability has provided us with more than hundred protein therapeutics and numerous other proteins/enzymes for various industrial applications and as diagnostics.. Echelard et al. ATryn was first approved as a therapeutic protein produced from transgenic animal (http://www. (j) Strasser et al.. no single biological system conforms to all these criteria (Rai & Padh. 1.. hGH (Human growth hormone). 1986. 2003. coli was the pioneer expression system in which first therapeutic protein. Protein synthesis is a tightly regulated process involving many enzymes and co-factors at various steps. (c) Sorensen & Mortensenl. 1..2.1.. 1977. 2004) and transgenic animals (Simons et al. 2005. Production of a protein outside of its natural host system is called heterologous protein production and the protein is termed as heterologous protein (Mahmoud. proteins used for therapeutic purposes constitute a special class with the stringent quality standards and therefore demand high value. 2006.. Needless to say that. 2003. yeast (Hitzeman et al.428 P. (i) Barta et al. (iii) be easy to handle and maintain. (m) Echelard et al. Desai et al. Proteins expressed in transgenic plants and animals have reached to various clinical and preclinical stages (Ma et al. (iv) be safe and economic. somatostatin. Donald and Jarvis. 2006). HBsAg (Hepatitis B surface antigen). Walsh. insects (Smith et al. 2001). 2001). (b) Pillai & Panchagnula. 2003). 1988. (k) Ma et al. A valuable expression system should (i) be capable of producing the required protein with right conformation. over a period of time a repertoire of need based expression systems have been developed as illustrated in Fig. Among these proteins. coli expression system to produce more complex therapeutic proteins (Sorensen and Mortensen.. 2005. 1 E. 2005. Wurm. Likewise other expression systems like mammalian cells (Moreira..N.. (d) Hitzeman et al. 1977). 2005). However. 2001. E. transgenic plants (Barta et al. 2001).

prokaryotic cells can be used only for proteins that do not require post-translational changes for biological activity. the issue is still regarded as a bottleneck in bringing protein based therapeutics to the public. one of which is now undergoing phase-I clinical trials (McCormick et al. Around 165 protein drugs are currently approved for human therapy and many more are in the clinical pipeline (Walsh. tissues or organisms are available as expression hosts. Desai et al. plants offer an attractive alternative (Boehm. precise transfer and integration of gene of interest. It can be achieved by the vacuum infiltration of leaves with recombinant A. whole plant or plant cell suspension culture as expression system. plants have been looked upon as a potential host system for expression of heterologous proteins. High levels of protein expression are achieved for a short time.. transgenics. This method has unique advantages like simplicity. 2005). while emphasizing its benefits and limitations. 2002). Hence plants in general provide a safer alternative to be used as a host system for production of desired proteins. Current demand of protein therapeutics exceeds its industrial production capacity and even after the increase in manufacturing capacity.. 1998). Virus infected plants have been used to produce several pharmaceutical proteins.N. transient or stable expression. eukaryotic systems are capable of a variety of post-translational modifications. Production of proteins in plants can be greatly increased to an industrial scale by cultivation of more plants. The nuclear transformation is generally achieved by using soil born pathogen Agrobacterium tumefaciens. In this review. and the fact that more than one vector can be used in the same plant. 2004). like intended use. The advantages of chloroplast transformation are many: the transgene copy number is high because of many chloroplasts in a single photosynthetic cell. there is no gene silencing. the systemic spread of the virus so that recombinant protein is produced in every cell. Commercial demand of therapeutic proteins The human genome project has identified a number of additional proteins having therapeutic potential. inflammatory diseases and anti-infective therapy. the recombinant proteins accumulate within the . cost. The chloroplasts transgenic system is another promising variation. The advantages of virus based production include the rapid onset of expression. 2003). it has a major disadvantage in terms of production time-scales which is being addressed by the development of alternative plant based production technologies. tumefaciens is the most commonly used vector for the transformation of a variety of dicots and a number of monocots (Lessard et al. 2007).. 2006). but are expensive to operate and have longer growth period (Yin et al. A. 2. The review also analyzes the strategies to maximize the expression of desired proteins in plant systems to overcome the major limiting steps in using plant systems for commercial production of therapeutic proteins. particularly therapeutic proteins. tumefaciens has a naturally occurring plasmid. These drugs are mainly used for cancer. 2003). tremendous progress has been made in this field and it has reached to the level where it could offer competitive alternative to the established production technologies that use bacteria.1. The cost of the proteins produced by plants is much lower than those expressed in mammalian cell lines (Evangelista et al. resulting in the transient transformation of many cells.up capacity Expression level Glycosylation Contamination risks Storage cost Bacteria Yeast Insect cell culture Mammalian cell culture Transgenic animal Plant cell culture Transgenic Plants Low Medium High High High Low Very low Short Medium Medium Long Very long Short Long High High Medium Very low Low High Very high High Low–high Low–high Low–moderate Moderate–high Moderate–high Moderate–high Absence Higher manosylation Higher manosylation similar to human Similar to human Minor differences Minor differences Endotoxins Low risk High Viruses. A variety of prokaryotic and eukaryotic cells. Another emerging transient-expression technology is based on the use of plant viruses as expression vectors. In the last decade. Although nuclear gene transfer is now a routine in many species. tumefaciens. Prions Low risk Low risk Moderate Moderate Expensive Expensive Expensive Inexpensive Inexpensive Note: Glycosylation pattern comparison is with human counterpart. 2.3.. Prokaryotic host systems are.P. Each of these expression systems has its own advantages and limitations some of which are listed in Table 1. System Production cost Production time scale Scale. On the other hand. stable transformation with single-copy insertion. the appropriate host can be selected. A portion of the T-DNA can be replaced by a gene of interest without affecting its transformation ability and is the molecular basis for almost all Agrobacteriummediated genetic transformation protocols (Lorence & Verpoorte. 2007). / Biotechnology Advances 28 (2010) 427–435 429 Table 1 Comparison of available expression hosts for the production of heterologous proteins.com/products/atryn. Each system offers some unique advantages making it an expression system of choice for a given protein. Based on particular requirements. 1998). etc. we have focused on use of plants as a host system for heterologous protein production. Plants as an expression system Plants have been explored and used as a source of food and medicine since ancient times and are generally considered to be safe for human use..html). The majority of plant-derived therapeutic proteins have been produced by nuclear transformation and the expressed proteins have been isolated and purified from the stable regenerated transgenic plants. Ti plasmid. callus. which is generally used for the expression of heterologous proteins which do not require complex post-translational modification. To fulfill the huge market demand with cost effective technologies. which has the ability to integrate its T-DNA and transform host cells (Escobar & Dandekar. Prions Viruses. reasonably low incidence of transgene silencing and the ability to transfer long stretches of T-DNA (N150 kb) (Veluthambi et al.. Therefore. Current manufacturing capacity for therapeutic proteins by traditional ways like fermentation and mammalian cell culture may not meet the market demand. Once these drug candidates are approved for the clinical use. in general easy to use and maintain as well as economical but are incapable of carrying out post-translational modifications. the demand for each drug will increase and high volume low cost production will become a necessity. Transplastomic plants are generated by introducing DNA into the chloroplast genome usually by particle bombardment. multiple genes can be expressed simultaneously like in operons of prokaryotes. allowing multimeric proteins to be assembled (Verch et al. including vaccine candidates and antibodies. Existing plant-based technologies There are different approaches for the expression of the heterologous proteins in plants: nuclear or plastid transformed. An attenuated A. To overcome many of these limitations.. 1. insect and cultured mammalian cells (Table 1). 2003). but generally the technique is insufficient for commercial-scale production (Gleba et al. Transient expression is generally used to verify transformation construct activity and to validate small amounts of recombinant protein. desired physicochemical characteristic of the expressed proteins.

. 2004 . apoplast (hEGF) was obtained in tobacco Allen et al. Thus. 2002). 1990 Gallie. III.2. Agrobacterium-mediated transformation is a method of choice in which gene of interest gets integrated with host nuclear genome (Ma et al. From all the available expression technologies. Proteins can be expressed in the edible part of the plant and can be consumed raw as an edible vaccine eliminating the need for downstream processes. low level of accumulation of expressed protein (Daniell et al. Transformation and integration: Attachment of SAR to DNA Increased expression of GUS gene in tobacco cells up to 140-fold 2. in the later part of the review we have focused the strategies based on the nuclear transformation techniques. nuclear transformation techniques are prominently used for the therapeutic protein production because of its unique advantages like stable expression of transgene and post-translational modifications. 1990 Mascarenhas et al. of CaMV GUS gene expression was 35S and mannopine synthetase increased 3–5 fold in leaves and 10–15 fold in hypocotyls and roots in tobacco and tomato plant..2) xylose which are absent in mammalian systems. Industrial scale production can be achieved by cultivation of more plants. because plants are not hosts for human infectious agents (Giddings et al. II. 2005). Therefore. More than 20 pharmaceutical proteins have been produced in plant cell-suspension cultures. / Biotechnology Advances 28 (2010) 427–435 chloroplast thus limiting toxicity to the host plant. bioactivity and favorable pharmacokinetics (Faye & Gomord. and have α-(1. and the absence of functional chloroplast DNA in the pollen of most crops provides natural transgene containment (Daniell et al. e. Factors influencing the heterologous protein production in plants Recent studies show that use of plant for production of recombinant proteins offers considerable advantages over the conventional systems: I. where they are more stable and offer ease in transportation without refrigeration.. affect its pharmacokinetic properties and create certain regulatory issues (Sethuraman & Stadheim. Advantages of plant expression systems 3. VII. Proteins produced by plants lack the terminal galactose and sialic acid residues commonly found in animals. are less likely to be contaminated with human pathogenic microorganisms than those derived from animal cells. although this depends on the protein of interest. erythropoietin. a sufficient dose of antigen. its expression must be sufficiently high to ensure efficiency. Desai et al. rabies vaccine expressed in tomato (Mason et al. tubers etc. only a few of these proteins have been expressed at yields sufficient for commercial production. Addition of Intron 2 and 6 of Increase the expression of CAT maize alcohol dehydrogenase-1 (Chloramphenicol acetyl transferase) gene by 12-fold and 20-fold respectively in maize protoplasts 3. resp. The major limitations of this system are low level of protein expression. 2. Desired proteins can be expressed in various targeted cells or tissues like seeds. Plant-made humanized glycosylated therapeutic proteins have already been produced. IV. must be delivered in a quantity of plant tissue that can be practically ingested in a single serving. 2006). In plant research. Plant cell cultures can be used for the production of small molecule drugs. plant produced glycoproteins can lead to immunogenicity. the use of plant as an expression system for production of therapeutic proteins has not moved from developmental research to For stable expression of a complex therapeutic protein. particularly when protein is expressed in specific tissues like seeds (Seon et al.3. the efforts are being focused on to increase the level of expression of therapeutic proteins (Streatfield. 2000).430 P.. 2002). 2003). Final yield or protein accumulation 104-fold higher Expression of Targeting to sub-cellular compartments human epidermal growth factor e. For proteins that are not extracted and purified prior to use. 2007). it is necessary to inhibit incorporation of some undesirable plant specific sugar molecules and to engineer new sugar molecules to obtain humanized glycoproteins.3) fucose and β-(1. 1994) Comai et al.. respective DNA sequence can be integrated into the plant nuclear DNA by means of suitable vector using appropriate transformation technologies. For an edible vaccine. Transcription Addition of Myb and leucine Increased expression zipper Hybrid promoter (Mac). Limitations of plant expression systems To date.. Unfortunately. 2004).g. which requires proper post-translational modifications. product yield and crop used (Twyman et al..N. It is an economical system as compared to mammalian cell culture and microbial fermentation. commercial productions. Apart from protein expression. whether purified or not. Ultimate expression of proteins is quite Table 2 Strategies used for enhancing expression of transgenes in plants.. the range of proteins that can be obtained in biologically active state from the chloroplast transformation is limited.. For the expression of required protein in plant.1% of the cost of mammalian cell cultures.. 1993 Perlak et al. tomato and rice cells.. Therapeutic proteins derived from plants. 2003). The only disadvantage from the prokaryotic character of plastids is its inability to do significant posttranslational modifications present in ER (endoplasmic reticulum). Plants can perform most of the post-translational modifications required for protein stability. VI.. 2003). Downstream processing when required is easy and less expensive. human granulocyte–macrophage colony stimulating factor (hGM-CSF) and hepatitis B antigen (Hellwing et al. 1. The main difference between the proteins produced by plant and animal is in sugar molecules attached to a protein. which is needed to confer protection. Tobacco suspension cells are the most popular system at present. In this system desired protein can be produced at 2–10% of the cost of microbial fermentation systems and at 0. but they are also advantageous for molecular farming because of the high level of containment that they offer and the possibility of producing proteins under current good manufacturing practice (cGMP) conditions. 2..g. increasing its stability for up to 3 years (Twyman et al. like the bacterial expression systems. 1994 (Hurst. and in tobacco hairy roots. The gene would then be transcribed and translated. However quality and quantity of proteins may be affected from lab scale to agriculture scale. and glycoengineering strategies that are based on the targeted expression of mammalian glycosyltransferases in transgenic plants have been developed (Sethuraman & Stadheim. including antibodies. 2004). 1996 Boulikas. although pharmaceutical proteins have also been produced in soybean. 1991 Wirth et al. Therefore. interleukins. accumulation level and stability of the final product are crucial parameters which affect the economics of protein production. 2006). 2001) and incorrect post-translational modifications of the protein.. To exploit plants for the production of therapeutic proteins. Translation Changing initiation codon context Increased expression GGUUUAUGU to CCUCCAUGU Codon optimization in tobacco Increase expression of cryIA (b) and tomato plants (Bacillus thuringiensis) up to 100fold 4. V..

1994. 2001 . avoiding repetitive homologous sequences. 2004 Shirasawa-Seo et al. An ideal plant expression vector should have (1) A wide selection of cloning sites. generally constitutive promoters give higher expression compared to strong tissue specific promoters. 1996 Frey et al. The expression levels Table 3 Most frequently used promoters in plant. a good expression vector is required as a vehicle to integrate the foreign DNA into the plant genome. Resulting expression of proteins in other than a target tissue can create metabolic burden on the host system and make downstream process difficult. co-factors.1. 2005 Christensen et al. 2002). different base composition between foreign DNA and the integration site. selection and screening for stable foreign DNA expression and developing site specific recombination systems. 1992 An et al.N.1. Several promoters of plant origin as well as plant pathogen origin have been developed and are listed in Table 3. The choice of the promoter depends upon the type of the protein produced. pMON.3. 1989 De Jaeger et al. few steps have been identified as rate limiting which can be improved to increase expression of a desired transgene (see Table 2).. 1985 Verdaguer et al. To alleviate some of this unpredictable gene silencing events an integrated approach may be used. combining a single-copy integration method with SAR (Scaffold attachment region) flanking may dramatically reduce the likelihood of homology dependent gene silencing (see Section 3. 1986 Van Haaren & Houck. Transcription can be controlled at the level of transcription initiation.. one should select transformed lines that are stable over many generations 431 rather than selecting high-expressing lines. (3) Small size. to minimize toxicity of therapeutic protein in host. The ability to increase transcription in targeted tissues and at a desired time has a practical application in the production of therapeutic proteins. Promoters are generally divided into three categories: constitutive. a good expression vector is required. This phenomenon may be less common in the case of constitutive promoters of plant origin such as ubiquitin and actin (Lessard et al. pGPVT. pBIN19. A common feature of foreign gene inactivation of most of genes is the presence of repeated homologous sequences known as HDGS (Homology Dependent Gene Silencing) (Meyer & Saedler.. 2001 De Veylder et al. Inactivation is generally observed with multiple copy integration at one or more sites. flanking foreign DNA with scaffold attachment regions (SAR). 2000). Certain synthetic promoters are also developed for maximizing the expression level. 1993 Ali & Taylor. screening and selection for plants with single-copy integration. 1997 Shaw et al. In both cases. A further advantage of using tissue specific promoter is convenient downstream purification process. 1989).. expression of protein with the help of viral originated constitutive promoter can result in gene silencing via co-suppression. These promoters can regulate when and where the transcription is desired. regulators.. Transcription is an early regulatory step in production of recombinant protein.. Promoters Origin Type of promoter Reference CaMV 35S CVMV (Cassava vein mosaic virus) C1 Component 8 of milk vetch dwarf virus Polyubiquitin-1 Actin Pristinamycin-responsive In2-2 Nopaline synthase Mannopine synthase Arcelin 5I promoter Patatin GBSS Maize globulin-1 Zein D hordein Β-Conglycinin 2A1 1 Lhcb3 Virus Virus Cotton leaf curl multan virus Virus Maize Rice Virus Maize Agrobacteria Agrobacteria Common bean Potato Potato Maize Maize Barley Soybean Tomato Arabidopsis Constitutive Constitutive Constitutive Constitutive Constitutive Constitutive Inducible Inducible Inducible Inducible Seed specific Tuber specific Tuber specific Embryo specific Endosperm specific Endosperm specific Embryo specific Fruit specific Leaf specific Odell et al. 10 to 13 Kb instead of the original 250 kb. integration of foreign DNA into plant genome is a key step in maximizing the final yield of a protein. Transcription initiation 3.. tissue specific promoter offers an added advantage.... 1991 Visser et al.3. Desai et al. pCAMBIA... inducible and tissue specific. and over expression effects (Finnegan & McElroy. A suitable vector fulfilling the individual requirements can be selected from the series (Hellens et al. Promoters.2. coli. 2000 Chen et al. 1984 Langridge et al.. 2000). Therefore. 3. In many cases. Foreign DNA can be inserted directly in the plant cell by particle bombardment or by Agrobacterium-mediated transformation. Transcription Expression of foreign gene is frequently regulated at the level of transcription. Various strategies are prescribed for the prevention of foreign DNA inactivation at integration level.6) (Allen et al. pGreen. 1997 Horvath et al. 1996). 1991 Belanger & Kriz. 3. Hurst.g. For commercial production of therapeutic proteins in plants. Moreover. pJJ. / Biotechnology Advances 28 (2010) 427–435 complex process carried out by many enzymes.3. In recent years. Integration of foreign gene After transformation. Choice of expression vectors For expression of therapeutic proteins in plant. The regulation of protein synthesis in plants could decrease production cost and provide qualitative advantages by deciding when and where the protein is being synthesized (Benfey & Chua.. It was observed that addition of trans-acting factors like Myb (binds to DNA and regulates the gene expression) and leucine zippers can boost the transcription (Boulikas. For example. (2) Broad host range ori (origin of replication) as well as ori of high copy number plasmid (for e. pGA482. 3. 1994). 1996).1..P.. and compartmentalized in several sub-cellular organelles. 2002 Liu et al. 1994). (4) Good choice of selectable markers for both bacteria and plants and (5) High transformation efficiency (Hellens et al. Col E1 from pBR322) to allow high yielding DNA preparations in E. 1991 Russell & Fromn. transgenes undergo inactivation resulting in loss of expression.. pC22. and so more efforts are made to increase the transcription level.. detrimental effects of sequences adjacent to the foreign DNA integration site. pNFHK and pPZP. RNA processing and RNA stability. and it is generally assumed that a high rate of the transcription can give a higher protein yield..1. 1996 Xie et al. 3. Numbers of optimized vectors are available commercially like pBECKS2000.

coli and yeast have demonstrated that rare codons and level of tRNA can affect translation time. 1989). 2005). Site directed mutagenesis is used to remove the rare codon by simply replacing a single base. In order to boost the translation. United States) uses a tomato hydroxyl-3-methylglutaryl CoA reductase 2 (HMGR2) promoter.1. / Biotechnology Advances 28 (2010) 427–435 achieved in the seeds of monocots using seed-specific promoters are higher than those using constitutive promoters. By using this strategy.N.3. Codon usage. Experiments with tobacco. Any part of this process. Initiation in eukaryotes is thought to follow a scanning mechanism whereby the 40S ribosomal subunit with co-factors (elF2. Optimizing at −5.1. Engineering the required sequence according to the codon usage can greatly increase the protein production and decrease the overall cost of the protein production. Synthetic promoters have also been developed that combine the most active sequences of multiple well-characterized natural promoters.. Several strategies have been followed to further boost transcription over that achieved with plant or plant viral promoters..2. AUUUA repeat is known to destabilize the transcript (Gutierrez et al. which is inducible by mechanical stress. elements of the cauliflower mosaic virus 35S promoter and the Agrobacterium Ti plasmid mannopine synthetase promoter have been combined where the hybrid synthetic promoter is several-folds more active than either component (Comai et al.expressed green fluorescent protein have demonstrated the benefit of codon optimization in plants (Rouwendal et al.2. tissue specific promoters are the primary choice. RNA stability is a critical factor to achieve a high level of expression. In certain cases inducible promoters are also used which can give the expression only in the presence of specific inducer. 3. Addition of the scaffold attachment sequence or matrix attachment regions next to promoters can further boost the expression (Allen et al. Consensus sequence in plants around the initiating AUG. Intron mediated enhancement. 2004).2. 2009).4.. Interestingly. is site directed mutagenesis.. 1990). expression of the Bacillus thuringiensis cryIA (b) (the insect control protein gene) in transgenic tobacco and tomato increased up to 100-fold (Perlak et al. 3.1. such as cauliflower mosaic virus 35S. 1997). Translation initiation Translational efficiency is thought to be controlled primarily by the rate of initiation. 1990). 1992). intron 1 of the maize sucrose synthase gene and intron 1 of the maize alcohol dehydrogenase gene (Lessard et al.. observed that the presence of a poly (A) tail on the 3′ end of mRNA greatly increased its translational efficiency. There are specific recognition sites that shorten the RNA half-life (Sullivan & Green. although plant genomes are AU rich. Transgene expression is activated when harvested tobacco leaves are sheared during processing. can be stacked to boost expression (Guerineau et al. 3.3.. In the case of development of oral vaccine where edible parts of plants are directly used for immunization. The role played by introns in the expression of eukaryotic genes in vivo remains poorly defined. could limit the initiation rate. intron 1 of the maize ubiquitin gene.4. elF4C. −4.4. Liu & Xue. Preferred codon usage differs between monocots and dicots. Multiple copies of enhancer sequences from a highly active promoter. A large number of rare codons or stable secondary structure in an mRNA might cause pausing of the ribosome translocation resulting in premature termination of transcript.. another approach. the mechanical gene activation (MeGA) system that was developed by Cramer (Crop Tech Corp. The stacking of the transcription units typically increases expression over the use of a single unit.3. Polyadenylation sites also strongly influence the stability of the message and ultimately. However. 1991). which leads to the rapid induction of protein expression. Some of these intron sequences contribute positively to the expression level of heterologous gene compared to native gene. elF3. Virginia. To increase the accumulation of recombinant protein. extensive runs or localized concentration of specific codon should be avoided. Some of these sites have been identified and are thought to correspond to specific binding sites (Taylor & Green. For example. 2001). 2008).. 3. 3. RNA stability After the transcript is being synthesized. met tRNA and GTP) bind the 5′ cap of mRNA and then descend through the untranslated leader scanning for the first AUG codon (Kozak. The observation that preferred codons are recognized by tRNAs in greatest abundance and rare codons are recognized by tRNAs in lowest abundance has led to the suggestion that tRNA abundance and codon usage have co-evolved (Higgs & Ran. Desai et al. and allows for multiple proteins to be expressed (During et al. Elongation and termination Ribosome translocation during protein translation was thought to be taking place at a constant rate. Sequence downstream to stop codon is critical for processing and should include signals targeting the message for polyadenylation. 1993). In vitro splicing systems have generated a wealth of information on the biochemical details of the splicing process and an exon-shuffling role in evolution has been proposed (Miller & Waterhouse.. One of the approaches to increase the translation efficiency in a given host is to optimize the codon usage by changing the nucleotide sequence without changing the amino acid sequence to suit the respective host (Gustafssion et al. Stack units are tandem repeats of the same promoter and terminator or other elements to reduce the potential for recombination or silencing. Translation 3. any sequence which is located immediately around the translation start site should be modified to fit the consensus initiation sequence. 3. In general when mammalian genes are expressed in plants. the level of protein expression in plants (Lin et al. RNA processing RNA processing like capping. 2002). certain sequences like intron splice sites. Studies in E. Premature termination may result in message destabilization. when GGUUUAUGU was changed to CCUCCAUGU. Plant systems generally use AUG (92% of 79 higher plant genes) as an initiation codon (Joshi. For example. However. and it is greatly different even between nucleus and plastid of the same plants (Kawabe & Miyashita.1. while optimizing for codon usage. . where it can be translated. these sequences can boost expression when inserted as synthetic introns into transgenes. AACAAUGGC and UAAACAAUGGCU are different from that used by animals (CACCAUGG) (Lutcke et al. It may be that this RNA processing itself encourages the efficient translocation of RNA to cytosol..3. Alternative to synthesizing whole sequence. 1990). 1995). While revising sequence for codon optimization. splicing and polyadenylation can affect the expression levels of therapeutic proteins. 1999). 2000). the 5′ cap and the poly (A) tail act synergistically to increase translation (Gallie. the 3′ end of an mRNA can also have a large effect on the efficiency of translation. 1987). 1993). otherwise remaining silent. it may be necessary to identify them and modify or delete them.4. A variety of plant introns have been co-opted from their native contexts and used to drive higher expression of transgenes: intron 1 of the rice actin gene. Gallie et al.. message destabilizing sequences and transcript termination sequences need to be removed or modified. −2 and −1 to C residues when non-optimal bases were present in positions −3 and +4 resulted in higher translational efficiency. 2003. 1991). which can be used in a number of cases.. which is affected by the structure of the leader and the AUG context. For example.432 P. one should also be aware of its consequence on mRNA secondary structure. In addition. so that the corresponding tRNA does not become rate limiting. Introns are normally spliced out of eukaryotic messages as the newly transcribed pre-mRNAs are processed in the nucleus.. usually within 24 h. 1987). In certain cases stacking of the transcription units is used for boosting the expression. Although the proper mechanism is not clear it is observed that incorporating introns into transgenes has an enhancing effect on the gene expression (Mascarenhas et al. it resulted in substantial increase in the rate of translation (Gallie.

proper folding and assembly of foreign protein can occur in the ER (Nuttall et al. co-expression with either protease inhibitors or with their co-factors and subunits.3.4. the base immediately following the stop codon (Angenon et al. Organ specific expression. Certain ER retention signal sequences like KDEL and HDEL are used to target the protein synthesis in ER and minimize protein degradation. is a transcriptional event associated with increased promoter methylation that is meiotically heritable (De Wilde et al. Free ricin A-chain. Co-expression with antigen was shown to improve expression of antibody (Stoger et al.. whereas co-expression of both A and B chains resulted in the formation of a disulfide-linked heterodimer. One of the important issues to be addressed is lower level of expression of proteins in plants. 4.. 3. Ricin A-chain expressed without its partner B-chain was degraded in tobacco. 3. Summary and concluding remarks The increased commercial uses of recombinant proteins as therapeutic drugs as well as other industrial commodity have mandated the search for appropriate and inexpensive expression hosts.1. Improving heterologous protein accumulation in plants remains a major bottleneck affecting the utility of this otherwise excellent expression system.. ER retention has resulted in 10–100 fold greater expression than those obtained by secretory pathway (Hellwing et al. The plant cell ER contains very few proteases and provides a relatively protective environment. Strategies used to avoid proteases assault include organelle specific expression. 1997). 3.1. Hernandez-Pinzon et al. / Biotechnology Advances 28 (2010) 427–435 UAA and UGA are preferred stop codon in plants. The most striking aspect in the stop codon context is the avoidance of a C (6%) and the preference for an A (41%) in the +1 position. 2005). Waterhouse et al. which is known to have a stabilizing effect on human proteins. Proteolytic degradation of foreign proteins can be minimized by targeting protein synthesis in ER (endoplasmic reticulum) rather than the cytosol (Conrad & Fiedler. On the other hand. 2004).5. are more prone to proteolysis. 2000). The introduction of multiple copies can be reduced by use of Agrobacterium. 2000). 1999). However. 2001). Coexpression of protease inhibitors does not show any adverse effect on plant growth and development and increase the expression of foreign protein. It is yet unclear whether plants can glycosylate using sialic acid. presumably because of its glycosylation pattern (Stevens et al. TGS is based on promoter inactivation.1.5. 1997). Gene silencing is based on the presence of homologous sequence at the coding region and it is a posttranscriptional process that is meiotically reversible. which makes them more vulnerable to protease activities. Furthermore. The review analyzes steps limiting heterologous protein production in plants and suggests several important avenues to improve expression of heterologous proteins that include: choice of vectors. Silencing seems to be positively correlated with the level of expression and copy number of the transgenes.. integration and stability of transgene. Use of tissue specific promoters or weak constitutive promoters can reduce the problem of gene silencing (Depicker & Van Montagu. Because of the presence of molecular chaperones and stabilizing agents.. Proteins. Plant-based expression system can replace mammalian cell culture and microbial systems for large scale production of recombinant therapeutics. IgG1 antibody produced by mouse hybridoma cells was more stable in tobacco leaf extract than the same antibody produced in plants. 1995). codon optimization and translation.6. Gene silencing Gene silencing is a potential problem during the expression of heterologous proteins in plants. 2002) perhaps by stabilization of antibody with antigen binding. 3. The low phenolic content of seeds and the presence of only a limited set of storage proteins are additional factors that can simplify downstream processing during product purification (Kusnadi et al. Co-expression of foreign protein with protease inhibitors is a promising strategy which can minimize the foreign protein degradation in plant tissues.. On the other hand. intracellular localization of expressed protein also determines its stability (Doran. transgenes present in multiple copies or expressed from strong constitutive promoters are more likely to be silenced. transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS). 2001). Addition of an N-termial histidine tag was also found to improve the stability of ricin B-chain in tobacco (Reed et al.2. Heterologous proteins produced by plants differ from their animal counterpart in terms of folding and higher order structure.. where plants have emerged as an attractive option for production of heterologous proteins. methods of transformation.e. 2006).. i. Such low protein yields are generally considered insufficient compared to other expression hosts.N. One of the best examples of this strategy is the development of transgenic rice cell suspension culture expressing recombinant human granulocyte–macrophage colony stimulating factor with co-expression of protease inhibitor sPI-II (Kim et al. 2001).5. Final yield or protein accumulation Low protein yield is a significant problem limiting the commercial exploitation of therapeutic protein production in plants. Most biopharmaceutical proteins accumulate in plant biomass at levels much lower than 1% of total soluble protein (Daniell et al. 1990).1. Expressing the protein in specific organs like seed or tuber can minimize degradation of foreign proteins. Protein which is synthesized in plant. proricin. 3.. Proteins which are not assembled with their co-factor and subunits are selectively degraded in plant cells (Callis.1. 2004). stability of transgene product and the last but not the least is proper glycosylation. A major function of proteases in plants is degradation of abnormal or incorrectly processed proteins. One of the best examples of this strategy is the expression of human epidermal growth factor in tobacco resulting in 104-fold higher yield (Wirth et al.. 2002). methylation and chromatin remodeling while PTGS is a sequence specific destruction of transcripts because of the presence of homologous interfering double stranded RNA (dsRNA) (Carthew. gene silencing. These organs contain endogenous protease inhibitors which offer increased protection against proteolytic attack..5. transcription and role of introns.. which are secreted into apoplast or extracellular space. Organelle specific expression. 433 3. 1998). Co-expression with co-factor and subunits. Two types of gene silencing have been observed in plants. This probably works by preventing formation of antisense transcripts by transcription from flanking endogenous genes into the transgenes (Ulker et al. 2001.. can be degraded by proteases.. certain technical advances need to be made before plants can be the expression system of choice. 2003). Co-expression with protease inhibitors. 3. which tends to result in fewer copies of transgenes than biolistic transformation (Dai et al. Desai et al. Flanking transgenes with matrix attachment region (MAR) sequences has been shown to reduce the occurrence of gene silencing. DNA methylation is often correlated with gene inactivation but the cause or the effect of gene silencing is not very clear (Veluthambi et al.. copy number. Proteases Proteases are the major factor responsible for lower accumulation of heterologous proteins.1. when not properly folded or synthesized. 2008). recent advances in genetic modification of glycosylating enzymes in plants have .P. 2007. and native toxin have different half-lives when expressed in tobacco protoplasts (Frigerio et al...5.5. based on the presence of a homologous sequence at the promoter region. 1998). Viral suppressors of silencing have been used quite effectively to prevent or reverse PTGS (Anandalakshmi et al. 1998).. For example.

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