Plant production of foreign proteins- An overview 

Feb 28, 2024 | Media

Professor Ed Rybicki, Head of Bio Pharming Research Unit, University of Cape Town

Plant-produced proteins are at first sight an exciting and the same time a controversial concept: exciting because of the much-touted possibility of being able to produce vast amounts of complex proteins at low cost; controversial because of the perception of the possibilities of contamination of the food supply, the potential for development of immunological tolerance to orally-introduced or edible preparations, and in some people’s minds, of potential regulatory and production issues to overcome.   

The central justifications for development of the technology – the promises – have been that protein production in plants is safer and more affordable, as well as infinitely scalable; that plants can often be used to produce biologically active proteins far more easily and quickly than can bacteria or yeast.   

The types of plants and types of plant tissues used for protein production and other vaccine antigens include the following: 

  • leaf and stem tissues of tobaccos of various species and varieties, Arabidopsis thaliana, alfalfa, spinach, and potatoes;  
  • aquatic weeds such as Lemna spp. (duckweed);  
  • seeds of rice, beans, maize and tobacco;  
  • fruits like tomatoes and strawberries;  
  • root vegetables like carrots;  
  • single-cell cultures of the algae Chlorella and Chlamydomonas 
  • suspension cell cultures of tobacco and other plants;  
  • hairy root cultures derived from various plants via Agrobacterium rhizogenes transformation;  
  • transformed chloroplasts of a variety of plant species.   

Antigens may be expressed in the cytoplasm and remain there or be localised to any of the plant organelles or compartments (nucleus, mitochondria, chloroplasts, vacuole, endoplasmic reticulum or apoplast) by means of specific signal peptides (Fischer et al., 2004).  The magic number for protein yield for economic extraction is often given as 1% of total soluble protein; however, this is seldom reached. 

Production via transgenesis 

Transgenic plants represent a potentially stable and cheap propagation source; however, development and selection of a suitable transgenic line can take many months, and production at high yield is often not attainable or stable, often due to the phenomenon of post-transcriptional or siRNA-dependent gene silencing.  This may be triggered by high concentrations of any particular mRNA, and in any case is reset at meiosis, leading to both immediate and potential long-term production instability (Baulcombe, 2004, 2005).This notwithstanding, (Li et al., 2006) report that a transgenic potato line expressing a human rotavirus VP7 protein was stable over 50 generations and maintained immunogenicity when fed to mice, indicating the potential of the system.  However, this is a sole example, and the stability of vaccine expression in transgenic plants is an under-investigated phenomenon.   

While constitutive or “green leaf” expression is the easiest to engineer, it can lead to problems if the protein interferes with plant development, and it is often difficult to purify proteins away from leaf constituents such as pigments, alkaloids and polyphenols.  Perhaps the simplest commercial whole plant transgenic expression system is that using the common duckweed, Lemna: Biolex Therapeutics claims their “LEX SystemSM” allows rapid product development due to ease of regeneration and rapid growth.  They have produced over 35 proteins under GMP conditions, many of which could not be produced in any other system {Biolex Therapeutics, 2008}.  The system is apparently very well suited to monoclonal antibody production, as it is possible to engineer glycosylation relatively easily (Cox et al., 2006).  

Expression and accumulation of proteins in seeds – via seed-specific promoters – is generally seen as preferable to expression in green tissue, because of far easier purification and higher accumulation levels (Lamphear et al., 2002; Takaiwa et al., 2007; Yang et al., 1999).  Seed-stored proteins are also generally stable for long-term storage, even at room temperature, due to the drying process which accompanies seed maturation.  A disadvantage is that transgenic expression can only begin to be assessed at seed set, which can take many months of plant growth. 

Transgenic single-cell cultures – whether of microalgae or of suspension-cultured plant cell lines – offer the advantages over whole-plant systems of a high level of containment and the possibility of producing proteins in bioreactors under “good manufacturing practice” (GMP) conditions, as is currently the case with conventional fermentation or cell culture techniques.  However, yields are generally not high (Fischer, 2004). There is usually little perceived advantage in these systems over animal or human or especially yeast cell culture production systems, other than generally simpler culture media for cell lines, and the possibility of using simple ponds or flow-through transparent “reactors”, salts and sunlight for algae.  However, Protalix Therapeutics have claimed that the therapeutic enzyme glucocerebrosidase produced in cultured carrot cells has a significant advantage over conventionally produced protein made in CHO cells: it has terminal mannose residues on its complex glycans, which has to be chemically added to the CHO cell product, as it is vital for uptake by macrophages to combat a lysosomal storage disorder (Shaaltiel et al., 2007) 

 

Expression of proteins from transformed chloroplasts often gives high yields, and avoids many of the problems associated with nuclear transformation (Daniell, 2006; Daniell et al., 2005).  However, this system is often not suitable for glycosylated proteins or secreted proteins, given the lack of suitable eukaryote-type machinery in what is essentially an intracellular prokaryote expression system.  The transformation system is also difficult, as is selection, and there is not as wide a choice of plant types as for nuclear transformation. 

Transient expression systems 

Such systems – wherein whole plants or cells are induced to express foreign proteins by means of recombinant virus infection or other means – are rapidly gaining popularity as a means both of quickly exploring viable promoter-protein-localisation options, and for the actual production of recombinant proteins.  The first of these expression systems to see the light was a recombinant tobacco mosaic virus (TMV) with its capsid protein fused to a malarial peptide (Turpen et al., 1995); by the late 1990s a HIV-1 gp41 peptide fused to cowpea mosaic virus capsids had been tested in mice both parenterally and orally (Durrani et al., 1998). Similar expression systems – as in the fusion of antigenic peptides to plant virus CPs – include Alfalfa mosaic virus (AMV) and Potato virus X (PVX), among others (Gleba et al., 2007; Yusibov et al., 1999, 2006).  Other useful fusion partners for chimaeric protein expression include Escherichia coli heat labile enterotoxin (LT-B), Cholera vibrio toxin B subunit protein (CTB) and Clostridium thermocellum lichenase (LickM), the last of which is expressed via a TMV-based system (Chichester et al., 2007; Choi et al., 2005; Companjen et al., 2006). Whole gene expression in plants is also possible via recombinant TMV and other viral vectors: however, the choice of appropriate vectors is limited, as is the choice of host plants, and vectors are often unstable and do not express large proteins very well.   

Another important transient expression system is Agrobacterium tumefaciens-based.  This technique relies on the infiltration of whole plant tissue with a suspension of Agrobacterium: T-DNA is transferred to a very high proportion of cells, where it can integrate or remain as an episome, and in either case will express its payload.  This transient somatic transformation or “agroinfection” allows very high levels of expression without the uncertainties inherent in the regeneration and propagation of transgenic plants, and, like viral vectors, at a time of the experimenter’s choosing.  While early application of the technology was mainly in the study of plant-pathogen interactions and in the investigation of endogenous gene expression, by 1999 it was being used successfully to produce antibody molecules and was being touted as a major advance in plant expression technology (Fischer et al., 1999). Its main advantage lies in the fact that simultaneous expression of a large number of constructs can very rapidly be investigated, unlike the case with plant viruses, which can exclude each other from infected cells.  It is also possible to do large scale production via vacuum-mediated agroinfiltration (Fischer, 2004) 

A very promising transient expression technique is the “MagniFection” system of Icon Genetics: this uses agroinfiltration to systemically deliver a TMV-based transient expression vector, which significantly amplifies the mRNA expression level compared to agroinfiltration, while maintaining the advantage of the systemic delivery due to the latter (Gleba et al., 2005).  Another system gaining popularity is the use of transgenic or transiently infected plants or cell cultures which can constitutively or inducibly express geminivirus replicons, resulting in amplified gene expression(Hefferon & Fan, 2004; Palmer & Rybicki, 2001) 

The main experience gained from these various systems is that there is no reliable way of predicting: 

  • if any given host plant will work   
  • whether or not a given DNA sequence will express protein at a reasonable level  
  • if that the protein will be stable  
  • if it will assemble correctly  
  • if it will be antigenically appropriate for the purpose 

Thus, empirical determinations – generally using transient expression systems for the sake of speed and convenience – are the only safe way of determining whether a given antigen can be expressed in a given system, and immunogenicity and preferably efficacy trials are the only way to determine whether they may work.   

 

 

 

 

 

Optimisation of expression 

As many have discovered over twenty years, the process of taking a gene encoding a candidate vaccine protein and expressing it in plants at a level that is acceptable for economic production (e.g: >50 mg/kg for antibodies), is far from being a trivial process.  Indeed, in many cases yields have been very low, especially in transgenic plants, and proofs of efficacy have suffered as a result.  This is of concern for oral vaccination schemes, as experiments have shown that orders of magnitude more protein is required via oral intake than parenterally for the same level of immune response to the same protein, even with the use of potent adjuvants.  The landmark 2005 potato-produced HBsAg human clinical trial (Thanavala et al., 2005) is a case in point: while parenteral vaccination requires only 40 µg of HBsAg, oral boosting with three 100 g doses of potato containing HBsAg doses of around 1 mg was only partially effective.  In a direct comparison of oral vs. parenteral dosing with the same vaccine, it was determined that 10 µg/dose of insect cell-produced HPV VLPs with adjuvant were required to orally immunise mice for the same response elicited by injection of 1 µg (Gerber et al., 2001; Rose et al., n.d.).  Others have shown that oral administration of crude extracts of L1-expressing insect cells could induce neutralising antibodies and L1-specific cytotoxic T-lymphocytes, indicating that similar plant preparations might work – especially since yields of between 1 -3 g/kg have been obtained (see above).  It is worth noting that much higher levels of HBsAg expression have since been obtained (Huang et al., 2008),which may yet make the dream of an oral HBV vaccine a reality. 

 

As an example of an attempt at optimisation of expression of the cancer-associated human pathogen HPV-16 major capsid protein L1, which we had previously only managed to produce to a level of 40 µg/kg via TMV expression (Varsani et al., 2006) , our group first investigated the effects of changing the type of tobacco used for transgenic expression; then, by using agroinfiltration, the effects of using L1 genes with three different codon optimisations, and targeting the protein to cytoplasm, ER and chloroplasts (Maclean et al., 2007).  Simply changing host from N. tabacum cv. Xanthi to SR1 allowed a 100-fold increase in expression of the native viral gene (A Varsani, J Maclean, EP Rybicki, unpublished), to ~0.5 mg/kg.  Via agroinfiltration work, we reiterated a surprising earlier finding that a human codon-use optimised gene worked best (Biemelt et al., 2003) and a plant codon-optimised version least well, compared to the native gene sequence, despite using completely different optimisations than the other group.  The removal of 24 amino acids at the C-terminus of L1 – which dramatically improves expression in yeast and insect cells – had a negative effect in agroinfiltrated plants.  We also showed that chloroplast export allowed significantly greater protein accumulation than cytoplasmic – and that subsequent generation of transgenic SR1 tobacco with the relevant constructs reiterated the cytoplasmic vs. chloroplast accumulation differences, and increased by a factor of ~20-fold the previous best transgenic HPV-16 L1 yield, to around 500 mg/kg.  However, this was achieved only in the T1 generation of plants regenerated from transformed callus: expression in all subsequent generations was either much lower, or completely absent (J Maclean, M Koekemoer, EP Rybicki, unpublished).  This points up another problem with attempts to maximise constitutive nuclear gene transgenic expression: the fact that gene silencing may occur!  Thus, while high-level HPV-16 L1 protein has been achieved in plants, to levels that could allow oral vaccines to be made, it appears that this will be from agroinfiltrated plants or transplastomic plants rather than stably transformed conventional transgenics. 

 

The optimisation of relevant HIV antigen expression is also an instructive lesson.  While the “conventional” developers of HIV vaccines are utilising multi-antigen approaches, and focussing on whole Gag and Env proteins in particular, it seems from published literature to have proved impossible so far to express whole HIV Env gp160 protein – although expression of a SIV gp130 was reported in maize seed (Hood et al., 2003) – and very difficult to express more than portions of Gag at reasonable yield (Halsey et al., 2008).  While Tat protein has been produced at quite high yields in spinach and tomato (Karasev et al., 2005; Ramírez et al., 2007), this too is more a curiosity than a serious vaccine candidate in the global HIV vaccine hunt.  It is most heartening, therefore, that Scotti et al. (Scotti et al., 2009) have recently reported that it is possible to produce whole Pr55Gag protein, at levels up to 800 mg/kg, in transplastomic tobacco.  The fact that the protein assembled into VLPs like those produced in animal cell systems, and which are increasingly seen as potential HIV vaccines, means that a viable plant-derived HIV vaccine may finally be possible. 

 

While it may be possible to some extent to predict ways of increasing recombinant protein expression in plants, I suspect that, on the basis of our experience with proteins from HPVs, HIV proteins, antibody fragments, influenza virus HA proteins and genes from rota- and orbiviruses, and the documented experiences of others, that the process is completely empirical.  The automatic assumption that “plant codon usage” will be optimal is naïve; so too are most assumptions based on experience in other non-plant cell culture systems.  There is no one universally suitable host or production system, although the use of agroinfiltration and deconstructed plant viral vectors seems to be on the way to becoming an industry standard.  It is advisable to explore all intracellular organelle and export targeting options, but also plastid transformation, as plastid targeting may not predict expression in these organelles. 

 

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