Stable expression systems in plants refer to genetically modified plant lines that are engineered to produce high levels of a target protein over each life cycle of a crop. They offer several advantages over traditional expression systems, including scalability, cost-effectiveness, and the ability to produce complex proteins with post-translational modifications. While transient expression is most often used as a tool for verification than for commercial-scale production, stable expression is typically used for larger-scale production. Choosing the most suitable system requires the careful consideration of the specific advantages and disadvantages associated with each method and depends on the specific type of project.
Various applications of stable expression systems are being employed today for a variety of purposes, including but not limited to, the production of vaccines, antibodies, and enzymes. Furthermore, stable expression has been employed in agriculture on a large-scale to improve the productivity of crops and help them resist stress from the environment and disease. Stable expression is also being considered in research purposes as a way to study gene function and various aspects of molecular biology. Overall, stable expression systems pose many advantages over other forms of expression, and offer a promising method for the scalable and sustainable production of protein in plants.
There are two major types of stable expression:
Nuclear Expression:
Nuclear expression involves the delivery of a gene of interest into a plant cell, such that the transgene becomes incorporated into the nuclear DNA where it is transcribed into mRNA and translated into the protein of interest by cell machinery. Advantages associated with this system include high expression levels and the ability to produce molecules with post-translational modifications. This system is suitable for long-term, stable expression because the transgene fully integrates into the cell genome, where it can be passed onto daughter cells for continued expression.
Nuclear expression systems subject the gene of interest to cellular processes such as transcriptional regulation and post-transcriptional processing. Regulatory elements such as promoters, enhancers, and silencers manage transcription to ensure stability overtime. Post-transcriptional modification such as capping, splicing, polyadenylation, and glycosylation help to establish stability and proper functionality of the final protein product. It is noteworthy that there is always the possibility of the transgene being subject to gene silencing/low expression event(s) due to the random nature of integration amongst other factors. Optimized methods that employ the use of advanced techniques like CRISPR-Cas9 gene editing can help reduce some of the limitations associated with random integration and gene silencing.
Common delivery methods associated with this system of expression include and gene gun and agrobacterium-mediated delivery. Using the gene gun method, the gene of interest is attached to a microscopic particle that is fired with high speed towards plant tissue, allowing the particles to penetrate cell walls and enter the intracellular region to ultimately integrate into the nuclear genome. In agrobacterium-mediated transformation, the transgene is inserted into a plasmid which is then introduced into cells of Agrobacteria tumefaciens. This is a bacterium that naturally targets plant cells, so when it is allowed to infect plant tissues, the transgene is transferred into the plant cell where it can enter the cell nucleus. For complex therapeutic protein that require post-translational modifications, it is best to employ agrobacterium-mediated transformation involving full integration into the nuclear genome.
Chloroplast Expression:
Chloroplast expression involves integrating the gene of interest into the genome of the chloroplast, such that the introduced gene survives cell replication and division, and is passed down the germ-line to future plants. Chloroplast expression has multiple advantages over nuclear expression. One advantage is that the chloroplast genome contains ‘regions of space’ between functional genes, such that foreign genes may be easily inserted using a transgene cassette. This allows for greater precision and choice in where the gene of interest is inserted. This also decreases the likelihood of inserting a gene into a region that is poorly transcribed, leading to lower expression. Furthermore, there is no record of splicing occurring to remove foreign genes that have been inserted into the chloroplast genome, an occurrence which has been documented in nuclear expression.
Challenges associated with this method include the double membrane around the chloroplast and the lack of viruses or bacteria that naturally target the chloroplast for infection that could be used as hosts for the gene of interest. However, as research continues to be conducted to optimize methods, challenges such as these are being addressed. For example, the development of the gene gun became a critical tool used in this type of system to deliver the transgene. The gene gun works by blasting plant tissue with DNA-coated particles, typically of gold. Gene guns are helpful when delivering DNA to cells that are difficult to impede, especially through cell walls and double membranes, so that the DNA in the cell is free to integrate into the genome. The efficiency of the gene gun combined with the high number of copies of the genome found within every chloroplast, this method is likely to obtain a very high number of copies of the introduced gene.
An advantage and disadvantage of chloroplast translation is that glycosylation does not occur. This is a pro for eliminating a source of immunogenicity during the production of therapeutic proteins that do not require glycosylation. However, this is a disadvantage for the production of therapeutics that do require glycosylation as a post-translational modification such as for antibodies. Therefore, chloroplast expression systems are best suited for the production of proteins that do not require extensive post-translational modification. This expression system has been used for the successful production of vaccines and human proteins.
In conclusion, there are many ways in which stable systems may be optimized to improve the protein production in plants. Choice of promoter can significantly impact the yield of the final protein product. Designing the transgene to include introns, untranslated regions, or specific tag sequences may improve the stability of the mRNA product following transcription and the efficiency of translation. Further, choosing an appropriate host plant based on its known characteristics and a compatible mode of transgene delivery may lead to the most suitable expression for the specific purposes of the project. Also, employing the use of enhancer and suppressor regions may decrease the likelihood of the transgene being spliced out. Well-planned protocols that employ appropriate modifications and specifications that increase the likelihood of achieving high stability and yield of the final protein product will continue to evolve and gain more and more traction on a commercial scale.
