Heterologous proteins: In vivo production of protein is a very complex process, which also involves post-translational modifications of protein, required for its stability and biological activity: like glycosylation, phosphorylation, and proper folding. Protein synthesis is a tightly regulated process involving many enzymes and co-factors at various steps. Production of a protein outside of its natural host system is called heterologous protein production and the protein is termed as heterologous proteins. Heterologous proteins are divided into three major groups: therapeutic proteins or those used for clinical diagnosis, proteins used as reagents for research and study purposes, and those proteins with various industrial applications. Among these proteins, proteins used for therapeutic purposes constitute a special class with stringent quality standards and therefore demand high value.
Commercial demand of therapeutic proteins: The human genome project has identified a number of additional proteins having therapeutic potential. Around 165 protein drugs are currently approved for human therapy andmanymore are in the clinical pipeline. These drugs are mainly used for cancer, inflammatory diseases and anti-infective therapy. Once these drug candidates are approved for the clinical use, the demand for each drug will increase and high volume low cost production will become a necessity. Current demand of protein therapeutics exceeds its industrial production capacity and even after the increase in manufacturing capacity, the issue is still regarded as a bottleneck in bringing protein based therapeutics to the public. Current manufacturing capacity for therapeutic proteins by traditional ways like fermentation and mammalian cell culture may not meet the market demand.
Advantages of plant expression systems: Recent studies show that use of plant for production of recombinant proteins offers considerable advantages over the conventional systems:
I. It is an economical system as compared to mammalian cell culture and microbial fermentation. In this system desired protein can be produced at 2–10% of the cost of microbial fermentation systems and at 0.1% of the cost of mammalian cell cultures, although this depends on the protein of interest, product yield and crop us.
II. Desired proteins can be expressed in various targeted cells or tissues like seeds, tubers etc., where they are more stable and offer ease in transportation without refrigeration, increasing its stability for up to 3 years
III. Industrial scale production can be achieved by cultivation of more plants. However quality and quantity of proteins may be affected from lab scale to agriculture scale.
IV. Therapeutic proteins derived from plants, whether purified or not, are less likely to be contaminated with human pathogenic microorganisms than those derived from animal cells, because plants are not hosts for human infectious agents.
V. 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, e.g. rabies vaccine expressed in tomato VI. Downstream processing when required is easy and less expensive, particularly when protein is expressed in specific tissues like seeds.
VII. Plants can perform most of the post-translational modifications required for protein stability, bioactivity and favorable pharmacokinetics.
Factors influencing the heterologous protein production in plants:
1) Choice of expression vectors: For expression of therapeutic proteins in plant, a good expression vector is required as a vehicle to integrate the foreign DNA into the plant genome. Foreign DNA can be inserted directly in the plant cell by particle bombardment or by Agrobacterium-mediated transformation. In both cases, a good expression vector is required. Numbers of optimized vectors are available commercially like pBECKS2000, pBIN19.
2. Integration of foreign gene: After transformation, integration of foreign DNA into plant genome is a key step in maximizing the final yield of a protein. In many cases, transgenes undergo inactivation resulting in loss of expression. Inactivation is generally observed with multiple copy integration at one or more sites, different base composition between foreign DNA and the integration site, detrimental effects of sequences adjacent to the foreign DNA integration site, and over expression effects.
Expression of foreign gene is frequently regulated at the level of transcription, and it is generally assumed that a high rate of the transcription can give a higher protein yield. Transcription is an early regulatory step in production of recombinant protein, and so more efforts are made to increase the transcription level. 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. Transcription can be controlled at the level of transcription initiation, RNA processing and RNA stability.
3.1. Transcription initiation
3.1.1. Promoters: Several promoters of plant origin as well as plant pathogen origin have been developed like CamV35s,CVMC,C1,actin etc. These promoters can regulate when and where the transcription is desired. Promoters are generally divided into three categories: constitutive, inducible and tissue specific. Certain synthetic promoters are also developed for maximizing the expression level. The choice of the promoter depends upon the type of the protein produced; generally constitutive promoters give higher expression compared to strong tissue specific promoters.
RNA processing like capping, splicing and polyadenylation can affect the expression levels of therapeutic proteins. Sequence downstream to stop codon is critical for processing and should include signals targeting the message for polyadenylation. Polyadenylation sites also strongly influence the stability of the message and ultimately, the level of protein expression in plants.
3.4. Translation (Translation initiation)
Translational efficiency is thought to be controlled primarily by the rate of initiation. Initiation in eukaryotes is thought to follow a scanning mechanism whereby the 40S ribosomal subunit with co-factors (elF2, elF3, elF4C,met tRNAandGTP)bindthe5′ cap ofmRNA and then descend through the untranslated leader scanning for the first AUG codon (Kozak, 1989). Any part of this process, which is affected by the structure of the leader and theAUGcontext, could limit the initiation rate.
Elongation and termination: Ribosome translocation during protein translation was thought to be taking place at a constant rate. 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. Premature termination may result in message destabilization. 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, the base immediately following the stop codon.
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.
3.6. Gene silencing
Gene silencing is a potential problem during the expression of heterologous proteins in plants. Two types of gene silencing have been observed in plants; transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS). TGS is based on promoter inactivation, methylation and chromatin remodeling while PTGS is sequence-specific destruction of transcripts because of the presence of homologous interfering double-strand RNA (dsRNA). Silencing seems to be positively correlated with the level of expression and copy number of the transgenes, i.e. transgenes present in multiple copies or expressed from strong constitutive promoters are more likely to be silenced. The introduction of multiple copies can be reduced by use of Agrobacterium, which tends to result in fewer copies of transgenes than biolistic transformation). Gene silencing is based on the presence of homologous sequence at the coding region and it is a posttranscriptional process that is meiotically reversible. On the other hand, gene silencing, basedon the presence of a homologous sequence at the promoter region, is a transcriptional event associated with increased promoter methylation that is meiotically heritable.