Humans have found plants to be a reliable and unlimited supply of tools and resources that have expanded over thousands of years. Plants are a source of versatile, sustainable raw material that we still use for food, construction, clothing, pharmaceuticals and more recently for molecular farming; the plant production of highly valuable molecules, including enzymes, vaccines, antibodies or complex natural products. The advances in plant molecular biology and the recent discovery and progression of viral-based expression vectors have led to the development of industrial-scale plant-based recombinant protein expression.
Even though we do not look alike, humans and plants are not as different as you may think, at least on a cellular level. We both have eukaryotic cells and share the same distant ancestry. These similarities are translated into some cellular features that are common for both organisms, including the way proteins are synthesized. Like humans, plants transcribe DNA into RNA and are not only able to translate this RNA into the polypeptide chains and to fold them correctly, but are also able to introduce posttranslational modifications, which are key for delivering their intricate cellular roles. Plants are therefore an excellent choice to produce highly valuable proteins, including biosimilars to those that are produced in animal cells.
But how can we express our chosen protein in a plant?
The first step is transforming the plant with the DNA that encodes for the protein of interest, in a manner that will allow it to be correctly transcribed and translated inside the plant cell. This is what is called plant-based recombinant protein expression, a fundamental development of which has been the plant viral-based expression vector technologies that enable high-level expression of the target protein. Plant viruses cause diseases by reprograming extremely efficiently the cellular machinery of susceptible hosts to their own benefit. Plant viral-based expression vector technologies take advantage of this process by selecting specific sequences from the viral genome to induce the expression of the target protein in large quantities, instead of the viral proteins. Please note that in many cases these viral expression vectors do not express the full viral genome, so no viable viral particles are formed, meaning that there is no risk of disease transmission by this process.
At Leaf Expression Systems we use the pEAQ-HT (from Hyper Trans®) expression vector technology, developed in Professor George Lomonossoff’s Lab at the John Innes Centre in Norwich (UK). This highly efficient expression vector is based on a deconstructed RNA-2 of the CPMV virus, where the 5’ and 3’ UTRs are used to improve the protein expression 1, reaching with some constructs, protein expression yield of greater than 1.5g/kg of fresh weight. CPMV-HT based technology has been also used successfully for vaccine mass production by Medicago Inc. (Canada).
The plant transformation step is done using a soil-borne bacteria able to transfer DNA between kingdoms, Agrobacterium tumefaciens. During natural infection, Agrobacterium transfers DNA from the bacteria and inserts it into the plant DNA, which will be responsible for the ‘hijacking’ part of the plant cell machinery to provide itself with nutrients. This infection causes tumors in the infected plants, named crown gall disease. In the lab, we use an Agrobacterium strain that has been “disarmed”, it cannot cause disease because it does not have all of the genes required for full virulence anymore. But the lab strain still retains the ability to transform the plant cell with foreign DNA. The pEAQ-HT vector contains the sequences needed by the bacteria to transfer it and insert it into the plant cell DNA. Once the transformation has been done, the plants will start to produce our chosen protein(s).
Aside from plants, biosimilar proteins can also be produced in mammalian or insect cells. These systems are expensive, as they require complex growth media and highly technical manufacturing production in sterile conditions. Plants, however, are cheap to grow and cultivate and do not require HEPA filtered air environments. In addition, the risk of contamination with a human virus or pathogenic bacteria is very low in plants. Microbial and viral contamination is one of the main causes of batch loss in large-scale mammalian cell culture facilities and can be an extremely expensive problem. Moreover, it is possible to express in plant molecules that are toxic in other production systems.
Plants also allow for a fully linearly scalable protein production, where the number of plants infiltrated correlates with the amount of protein obtained. Finally, protein production is maximized between day 3 and 5 after inoculation with the bacteria enabling a very rapid turnaround time for production. The hand infiltration technique that we employ at the start of a project enables us to test the expression level and have an approximation of the protein yield in less than two weeks.
Plants have become not only a source of food or materials but also an excellent way of producing sustainably highly valuable molecules at reduced costs, making them the ideal choice for expressing your protein of interest.
If you would like any more information, be that more specific details of our process or about how we may be able to help you with a project, please do get in touch.
Dr Albor Dobon Alonso
Senior Molecular Biologist
1 Plant Biotechnol J. Sainsbury F, Thuenemann EC, Lomonossoff GP. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J. 2009;7:682–93.