Single Domain Antibody (sdAb) Expression in Plant
Plant System for sdAb Production
Transgenic plants for the production of high-value recombinant antibodies at high concentrations is emerging as a promising alternative method for conventional systems, such as mammalian cell expression systems and bacteria expression systems. This system can express sdAb fusion proteins with good binding ability and functionality, and they can do so in high quantity; Plant system-derived sdAbs are often used owing to their free of possible contamination with human pathogens and toxins. Beyond that, there are unique features associated with plants, such as convenience, long-term protein storage in seeds, capability of performing post-translational modifications, easy transformation, and capacity of delivery after production in some plant tissues.
Basic Strategies for Plant System
In general, there are three methods developed for expressing antibody fragments in plants, namely, transient transfection expression, stable transgenic expression, and plastid genome expression. The principal approach associated with sdAbs production involves transient and stable nuclear genome expression infiltrated by Agrobacterium strains. Because T-DNA integration into the host plant genome is imperative for transgene expression not only in large-scale transient expression but also for the production of stable transgenic plants. The Agrobacterium strains containing binary T-DNA vectors can be used for transgene delivery. The T-DNA region cloning the sequence of interest of these binary vectors is transferred from the Agrobacterium intermediate host via Agrobacterium infiltration into the nucleus of the plant cell and results in a high and rapid recombinant antibody production in the leaves. Transient expression using viral vectors could achieve a higher yield in some cases when compared with the yields observed in stably transformed plants.
Fig. 1 Plant-based transient expression system.1
Plant Hosts
Among various plant hosts, tobacco (Nicotiana benthamiana) is the most commonly used host for transient expression of sdAbs. This system allows the production of high yields sdAbs within a relatively short time after the expression plasmid has been obtained, and the average yields in this host are over 1% of all total soluble proteins (TSP). However, intensive upstream processing is required as each plant must be infected with Agrobacterium tumefaciens cells by vacuum infiltration. In addition, N. benthamiana is often used for the production of stable transgenic plants through a procedure that requires long development time.
Another plant, the Arabidopsis thaliana seed, is a more convenient vehicle to produce oral delivery sdAbs; these seeds show much higher yields in some cases when compared with N. benthamiana. The generation of stable A. thaliana transformants allows the production of scalable amounts of VHH-Fc antibodies in seeds within a year. Furthermore, other plant production systems, such as rice seeds and nuclear transgenic potatoes, are also described for sdAbs thus far.
Brief Workflow for sdAb Production
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The production via transient expression in N. benthamiana leaves
- Preparation of Agrobacterium strains containing binary T-DNA vectors
- Infiltration of an Agrobacterium suspension in N. benthamiana leaves
- Harvest of the infiltrated N. benthamiana leaves and protein extraction
- Determination of the antibody concentration in the leaf extracts
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The production in A. thaliana seeds
- A. thaliana floral dip transformation
- Protein extraction from A. thaliana seeds
- Determination of the antibody concentration in seed extracts
- Western blot analysis to evaluate the accumulation level of antibodies
Advantages of Plant System
The plant system offers many practical, economical, and safety advantages over other systems, such as the non-risk for contamination with viruses, pathogens and toxins, unlimited potential for production upscaling, and the low production cost. The recombinant antibodies produced in plants can be assembled correctly and harbor posttranslational modifications. Additionally, plant cell cultures, as well as transgenic plants, have been used as promising tissues because of their stable environment to protect recombinant antibodies against degradation. Therefore, the plant system might be an economical alternative to produce extremely large amounts of sdAbs.
Reference
- Sainsbury, Frank, and George P. Lomonossoff. "Transient expressions of synthetic biology in plants." Current opinion in plant biology 19 (2014): 1-7. Distributed under Open Access license CC BY 3.0, without modification.
Case Study
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