The development of genetic engineering and cloning created many possibilities for the expression and isolation of heterologous proteins for research and biopharmaceutical purposes. By the end of the last century, several protein expression systems had been developed in a variety of media including bacteria, yeast, insects, mammalian systems and plants.
Recent technological advances now enable protein expression on a larger scale, opening up the commercial possibilities for enzyme, antibody and vaccine production. In such cases, the system in which the protein is expressed must be easy to culture and maintain, grow rapidly and produce large amounts of protein.
Plant-based expression systems have a range of technical advantages including insertion gene size, yield, propagation, protein assembly and folding accuracy, product quality and homogeneity, distribution and storage temperature, together with scale-up speed and flexibility, leading to cost savings in production and storage of the product.
Leaf Expression Systems produces proteins and complex natural products using Hypertrans®, a novel proprietary transient plant expression system which is efficient, safe and simple to use.
Hypertrans® uses the non-cultivated tobacco species, N. Benthamiana, as the bioreactor. This plant is very fast-growing, with hundreds of plants capable of yielding product in as little as one week. The plants are grown on-site in a controlled containment facility, providing quality assurance and security.
Traditional virus-based vectors generally rely on replication of the virus genome to achieve high levels of protein. However, this has a number of disadvantages in terms of the size and complexity of the proteins which can be expressed, together with containment issues. The Hypertrans® system relies on making mRNAs “hyper-translatable” by flanking them with modified untranslated regions from the deleted version of Cowpea mosaic virus RNA-2 (CPMV-HT). This permits the extremely high-level and rapid production of proteins without viral replication.
To exploit the high levels of expression achieved with CPMV-HT, a series of small proprietary modular binary vectors, known as pEAQ vectors, are incorporated within the system. Non-essential sequence has been removed from the original vectors and unique restriction sites introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days.
Furthermore, these vectors allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C- terminal histidine tags may be fused to the target sequence as required. These vectors produce an easy and quick tool for the production of milligram quantities of recombinant proteins from plants with standard plant research techniques at a bench-top scale.
Scale-up and Production
Recombinant Agrobacterium tumefaciens containing CPMV-HT within the proprietary pEAQ modular vectors is introduced into the gaps between leaf cells by vacuum infiltration, resulting in rapid transient expression of proteins. The process usually reaches a maximum yield after about a week.
The flexible nature of the system enables production to be scaled from small research quantities to pilot plant scale production very quickly. The speed of the system means that it can rapidly produce large amounts of protein and so it is well suited to rapidly responding to emergencies like pandemics.
Hypertrans® is currently used in the production of:
Complex proteins such as antibodies or enzymes for research applications
Antigens including virus-like particles for vaccine development or nanotechnology
Metabolites for metabolic pathway engineering to generate complex biochemicals that are difficult or impossible to synthesise chemically or are only found in rare species or trace amounts in nature.