Expression of the VP1 antigen from foot-and-mouth disease virus in a bacterial and plant-based expression system

The suitability of a plant-based transient expression system using the agro-infiltration technique was compared to an Escherichia coli (E. coli)-based expression system to produce the VP1 protein from Serotype O, South Korean strain, of the foot-and mouth disease virus (FMDV). The full-length VP1 coding sequence was expressed in Escherichia coli as a fusion protein and purified as a His-tagged VP1 fusion protein with a yield of 14 mg L-1 bacterial culture. For transient expression in tobacco, the VP1 coding sequence was cloned into binary vector pMYV497, containing a CTB (cholera toxin B subunit) signal peptide and SEKDEL ER retention signal, and transiently agro-infiltrated into non-transgenic N. benthamiana and transgenic N. tabacum plants constitutively expressing the rice cysteine protease inhibitor OC-I. A protein resembling VP1 was detected using immuno-blotting analysis in both N. benthamiana and OC-I N. tabacum plants seven days post agro-infiltration. Although a possible stabilizing effect on VP1 was found due to OC-I expression, protein yields were not significantly different between transformed OC-I and non-OC-I control plants. Also, simultaneous co-infiltration with a plasmid allowing additional transient OC-I expression did not significantly improve VP1 production. The average VP1 amount achieved in OC-I expressing plants was 0.75% of total soluble protein. Overall, this study has shown that transient VP1 expression in tobacco is possible, but requiring further optimization, and that OC-I might have a stabilizing effect against proteolytic degradation of VP1 during advanced stages of senescence in agro-infiltrated plants coinciding with peaks in protein expression. CopyrightDissertation (MSc)--University of Pretoria, 2012.Plant Scienceunrestricte

Loop replacement design : a new way to improve potency of plant cystatins

Comment on https://doi.org/10.1111/febs.16288Plant cystatins function as competitive inhibitors of cysteine proteases. Similar to other defence proteins, cystatins include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Protein engineering approaches, such as point mutations, at these functionally relevant amino acid sites have already been found to be a powerful tool in improving the inhibitory properties of cystatins. Such engineered cystatins not only better protect against digestive proteases of herbivorous arthropods but also against cysteine proteases of several other plant pests as well as against cysteine proteases produced in plant during stress-induced senescence. Despite previous engineering successes, an urgent need still exists to further improve both plant cystatin potency and specificity. Tremblay and colleagues propose in this issue a new cystatin engineering strategy to substitute the function-related structural elements (SEs) of a cystatin by the corresponding elements of an alternative cystatin. This strategy, possibly combined with direct cystatin gene editing in a target plant, might provide an innovative way to control cysteine protease activity.NRF incentive funding.https://febs.onlinelibrary.wiley.com/journal/17424658hj2023Forestry and Agricultural Biotechnology Institute (FABI)Plant Production and Soil Scienc

Proteolysis of recombinant proteins in bioengineered plant cells

Plants are increasingly used as alternative expression hosts for the production of recombinant proteins offering many advantages including higher biomass and the ability to perform posttranslational modifications on complex proteins. Key challenges for optimized accumulation of recombinant proteins in a plant system still remain, including endogenous plant proteolytic activity, which may severely compromise recombinant protein stability. Several strategies have recently been applied to improve protein stability by limiting protease action such as recombinant protein production in various sub-cellular compartments or application of protease inhibitors to limit protease action. A short update on the current strategies applied is provided here, with particular focus on sub-cellular sites previously selected for recombinant protein production and the co-expression of protease inhibitors to limit protease activity.http://www.tandfonline.com/loi/kbie20hb201

Agroinfiltration contributes to VP1 recombinant protein degradation

There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modelling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modelling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.The National Research Foundation (NRF) and the Genomics Research Institute (GRI), South Africa as well as NRF incentive funding to Karl Kunert and a NRF bursary to Priyen Pillay.http://www.tandfonline.com/loi/kbie202017-08-31hb2016Plant Scienc

Review : The future of cystatin engineering

Plant cystatins are naturally occurring protease inhibitors that prevent proteolysis by papain-like cysteine proteases. Their protective action against environmental stresses has been relatively well characterised. Still, there is a need to greatly improve both potency and specificity based on the current rather poor performance of cystatins in biotechnological applications. Research in creating more potent and specific cystatins, including amino acid substitutions in either conserved cystatin motifs and/or at variable amino acid sites, is reviewed. Existing gaps for better understanding of cystatin-protease interactions are further explored. Current knowledge on multi-cystatins or hybrid protease inhibitors involving cystatins as an additional option for cystatin engineering is further outlined along with the nuances of how cystatins with rather unusual amino acid sequences might actually help in cystatin engineering. Finally, future opportunities for application of cystatins are highlighted which include applications in genetically modified transgenic plants for environmental stress protection and also as nutraceuticals, as part of more nutritious food. Further opportunities might also include the possible management of diseases and disorders, often associated with lifestyle changes, and the most immediate and promising application which is inclusion into plant-based recombinant protein production platforms.International Foundation of Science (IFS grant C/5151-1), the NRF Incentive funding for rated researchers (90779) and the NRF National Bioinformatics Functional Genomics program(86947). Funding received from the Genomic Research Institute (GRI), University of Pretoria.http://www.elsevier.com/locate/plantsci2017-05-31hb2016Forestry and Agricultural Biotechnology Institute (FABI)Plant Production and Soil SciencePlant Scienc

Use of transgenic Oryzacystatin-I expressing plants enhances recombinant protein production

Plants are an effective and inexpensive host for the production of commercially interesting heterologous recombinant proteins. The Escherichia coli-derived glutathione reductase was transiently expressed as a recombinant model protein in the cytosol of tobacco plants using the technique of leaf agro-infiltration. Proteolytic cysteine protease activity progressively increased over time when glutathione reductase accumulated in leaves. Application of cysteine protease promoter–GUS fusions in transgenic tobacco identified a cysteine protease NtCP2 expressed in mature leaves and being stress responsive to be expressed as a consequence of agro-infiltration. Transgenic tobacco plants constitutively expressing the rice cysteine protease inhibitor oryzacystatin-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher glutathione reductase activity and also higher glutathione reductase amounts in transgenic plants. Overall, our work has demonstrated as a novel aspect that transgenic tobacco plants constitutively expressing an exogenous cysteine protease inhibitor have the potential for producing more recombinant protein which is very likely due to the reduced activity of endogenous cysteine protease.This work was supported by a grant from the National Research Foundation in South Africa.http://link.springer.com/journal/12010hb201

Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9

The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant 1XTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No dierences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and 1XTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.The Department of Science and Innovation (DSI), South African Medical Research Council– Strategic Health Innovation Partnership (SAMRC SHIP), National Research Foundation (NRF), Council for Scientific and Industrial Research (CSIR), and the CSIR: Young Researcher Establishment Fund.http://www.frontiersin.org/Plant_Sciencedm2022Plant Production and Soil Scienc

Engineering Approaches in Plant Molecular Farming for Global Health

Since the demonstration of the first plant-produced proteins of medical interest, there has been significant growth and interest in the field of plant molecular farming, with plants now being considered a viable production platform for vaccines. Despite this interest and development by a few biopharmaceutical companies, plant molecular farming is yet to be embraced by ‘big pharma’. The plant system offers a faster alternative, which is a potentially more cost-effective and scalable platform for the mass production of highly complex protein vaccines, owing to the high degree of similarity between the plant and mammalian secretory pathway. Here, we identify and address bottlenecks in the use of plants for vaccine manufacturing and discuss engineering approaches that demonstrate both the utility and versatility of the plant production system as a viable biomanufacturing platform for global health. Strategies for improving the yields and quality of plant-produced vaccines, as well as the incorporation of authentic posttranslational modifications that are essential to the functionality of these highly complex protein vaccines, will also be discussed. Case-by-case examples are considered for improving the production of functional protein-based vaccines. The combination of all these strategies provides a basis for the use of cutting-edge genome editing technology to create a general plant chassis with reduced host cell proteins, which is optimised for high-level protein production of vaccines with the correct posttranslational modifications

Engineering Approaches in Plant Molecular Farming for Global Health

Since the demonstration of the first plant-produced proteins of medical interest, there has been significant growth and interest in the field of plant molecular farming, with plants now being considered a viable production platform for vaccines. Despite this interest and development by a few biopharmaceutical companies, plant molecular farming is yet to be embraced by ‘big pharma’. The plant system offers a faster alternative, which is a potentially more cost-effective and scalable platform for the mass production of highly complex protein vaccines, owing to the high degree of similarity between the plant and mammalian secretory pathway. Here, we identify and address bottlenecks in the use of plants for vaccine manufacturing and discuss engineering approaches that demonstrate both the utility and versatility of the plant production system as a viable biomanufacturing platform for global health. Strategies for improving the yields and quality of plant-produced vaccines, as well as the incorporation of authentic posttranslational modifications that are essential to the functionality of these highly complex protein vaccines, will also be discussed. Case-by-case examples are considered for improving the production of functional protein-based vaccines. The combination of all these strategies provides a basis for the use of cutting-edge genome editing technology to create a general plant chassis with reduced host cell proteins, which is optimised for high-level protein production of vaccines with the correct posttranslational modifications

South Africa's indigenous microbial diversity for industrial applications: A review of the current status and opportunities

The unique metagenomic, metaviromic libraries and indigenous micro diversity within Southern Africa have the potential for global beneficiation in academia and industry. Microorganisms that flourish at high temperatures, adverse pH conditions, and high salinity are likely to have enzyme systems that function efficiently under those conditions. These attributes afford researchers and industries alternative approaches that could replace existing chemical processes. Thus, a better understanding of African microbial/genetic diversity is crucial for the development of “greener” industries. A concerted drive to exploit the potential locked in biological resources has been previously seen with companies such as Diversa Incorporated and Verenium (Badische Anilin-und SodaFabrik-BASF) both building business models that pioneered the production of high-performance specialty enzymes for a variety of different industrial applications. The market potential and accompanying industry offerings have not been fully exploited in South Africa, nor in Africa at large. Utilization of the continent's indigenous microbial repositories could create long-lasting, sustainable growth in various production sectors, providing economic growth in resource-poor regions. By bolstering local manufacture of high-value bio-based products, scientific and engineering discoveries have the potential to generate new industries which in turn would provide employment avenues for many skilled and unskilled laborers. The positive implications of this could play a role in altering the face of business markets on the continent from costly import-driven markets to income-generating export markets. This review focuses on identifying microbially diverse areas located in South Africa while providing a profile for all associated microbial/genetically derived libraries in this country. A comprehensive list of all the relevant researchers and potential key players is presented, mapping out existing research networks for the facilitation of collaboration. The overall aim of this review is to facilitate a coordinated journey of exploration, one which will hopefully realize the value that South Africa's microbial diversity has to offer