Production Of Recombinant Proteins In Plants Research Articles

Development of a Molasses-Based Medium for Agrobacterium tumefaciens Fermentation for Application in Plant-Based Recombinant Protein Production

The Agrobacterium-mediated transient gene expression system is a rapid and efficient method for heterologous recombinant protein expression in plants. The fermentation of genetically modified Agrobacterium tumefaciens is an important step in increasing the efficiency of recombinant protein production in plants. However, the limitation of this system that makes it economically non-competitive for industrial-scale applications is the Agrobacterium suspension production cost. In this study, the utilization of sugarcane molasses as an alternative low-cost source of carbon at a concentration of 8.7 g/L and nitrogen at a concentration of 2.4 g/L for Agrobacterium cultivation was investigated. Molasses pretreatment using sulfuric acid (SA) was applied before fermentation, and it resulted in a maximum specific growth rate of 0.232 ± 0.0063 h−1 in the A. tumefaciens EHA105 culture. The supplementation of antibiotics in the molasses-based medium was shown to be unnecessary for plasmid maintenance during fermentation in both Agrobacterium strains, which helped to reduce the production cost. We evaluated recombinant protein production using an Agrobacterium culture without antibiotic supplementation in the growth media by demonstrating green fluorescent protein expression in wild-type Nicotiana benthamiana leaves. In the evaluation of the culture medium cost, the molasses-based medium cost was 6.1 times lower than that of LB. Finally, this study demonstrated that the newly developed molasses-based medium for Agrobacterium fermentation is a feasible and effective medium for transient recombinant protein production in plant tissues.

Maximizing the Production of Recombinant Proteins in Plants: From Transcription to Protein Stability.

The production of therapeutic and industrial recombinant proteins in plants has advantages over established bacterial and mammalian systems in terms of cost, scalability, growth conditions, and product safety. In order to compete with these conventional expression systems, however, plant expression platforms must have additional economic advantages by demonstrating a high protein production yield with consistent quality. Over the past decades, important progress has been made in developing strategies to increase the yield of recombinant proteins in plants by enhancing their expression and reducing their degradation. Unlike bacterial and animal systems, plant expression systems can utilize not only cell cultures but also whole plants for the production of recombinant proteins. The development of viral vectors and chloroplast transformation has opened new strategies to drastically increase the yield of recombinant proteins from plants. The identification of promoters for strong, constitutive, and inducible promoters or the tissue-specific expression of transgenes allows for the production of recombinant proteins at high levels and for special purposes. Advances in the understanding of RNAi have led to effective strategies for reducing gene silencing and increasing recombinant protein production. An increased understanding of protein translation, quality control, trafficking, and degradation has also helped with the development of approaches to enhance the synthesis and stability of recombinant proteins in plants. In this review, we discuss the progress in understanding the processes that control the synthesis and degradation of gene transcripts and proteins, which underlie a variety of developed strategies aimed at maximizing recombinant protein production in plants.

Improving the production of plant-based recombinant protein: a review

Recombinant proteins provide new means for disease treatment, while creating considerable economic benefits. Using commercial crops (mainly tobacco), cereal crops, legumes, and vegetable crops to produce recombinant proteins with medicinal value is a hot-spot for research in "molecular farming". Although many recombinant proteins have been expressed in plants, only a small number have been successfully put into use. To overcome the problems that greatly hamper the development of recombinant protein production in plants, researchers have improved expression systems to increase the yield of recombinant proteins. Starting from analyzing the problems of low yield and/or low biological activity of recombinant proteins produced by plants, the optimization strategies to solve these problems were reviewed, and future research directions for improving the yield of recombinant proteins produced by plants were proposed.

The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants.

Plants have long been considered a cost-effective platform for recombinant production. A recently recognized additional advantage includes the low risk of contamination of human pathogens, such as viruses and bacterial endotoxins. Indeed, a great advance has been made in developing plants as a “factory” to produce recombinant proteins to use for biopharmaceutical purposes. However, there is still a need to develop new tools for recombinant protein production in plants. In this study, we provide data showing that the B1 domain of Streptococcal protein G (GB1) can be a multi-functional domain of recombinant proteins in plants. N-terminal fusion of the GB1 domain increased the expression level of various target proteins ranging from 1.3- to 3.1-fold at the protein level depending on the target proteins. GB1 fusion led to the stabilization of the fusion proteins. Furthermore, the direct detection of GB1-fusion proteins by the secondary anti-IgG antibody eliminated the use of the primary antibody for western blot analysis. Based on these data, we propose that the small GB1 domain can be used as a versatile tag for recombinant protein production in plants.

Principles and Applications of Recombinant Protein Production in Plants

Recombinant protein production has become a growing sector all around the world such as in therapeutic applications, pharmaceutical, agriculture etc. Although, a number of systems for protein expression have been developed and there is an increasing requirement for efficient methods of large-scale production. Therefore recombinant proteins are produced by various ways to generate large quantities for commercial and research applications in a different host such as in bacteria, yeasts, insects, and mammalian culture. Among these, plant systems are mostly preferred since they have a developed eukaryotic system. The expression of recombinant proteins in plants and plant cells has been promoted as an alternative cost-effective production platform. In this review we described, challenges and advantages of plants as expression systems for proteins and discussed unique advantages of producing proteins recombinantly in different plants; tobacco, rice, and maize.

Therapeutic recombinant protein production in plants: Challenges and opportunities

Societal Impact StatementTherapeutic protein production in plants is an area of great potential for increasing and improving the production of proteins for the treatment or prevention of disease in humans and other animals. There are a number of key benefits of this technique for scientists and society, as well as regulatory challenges that need to be overcome by policymakers. Increased public understanding of the costs and benefits of therapeutic protein production in plants will be instrumental in increasing the acceptance, and thus the medical and veterinary impact, of this approach.SummaryTherapeutic recombinant proteins are a powerful tool for combating many diseases which have previously been hard to treat. The most utilized expression systems are Chinese Hamster Ovary cells and Escherichia coli, but all available expression systems have strengths and weaknesses regarding development time, cost, protein size, yield, growth conditions, posttranslational modifications and regulatory approval. The plant industry is well established and growing and harvesting crops is easy and affordable using current infrastructure. Growth conditions are generally simple: sunlight, water, and the addition of cheap, available fertilizers. There are multiple options for plant expression systems, including species, genetic constructs and protein targeting, each best suited to a particular type of therapeutic protein production. Transient expression systems provide a mechanism to rapidly transfect plants and produce therapeutic protein in a matter of weeks, rather than the months it can take for many competing expression systems, while proteins targeted to cereal seeds can be harvested, stored and potentially purified much more easily than in competing systems. Current challenges for plant expression systems include a lack of regulatory approval, environmental containment concerns and nonhuman glycosylation, which may limit the scope of the type of therapeutic proteins that can be manufactured in plants. The specific strengths of plant expression systems could facilitate the production of certain therapeutic proteins quickly and cheaply in the near future.

Activity-based proteomics reveals nine target proteases for the recombinant protein-stabilizing inhibitor SlCYS8 in Nicotiana benthamiana.

SummaryCo‐expression of protease inhibitors like the tomato cystatin Sl CYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity‐based protein profiling to identify proteases that are inhibited by Sl CYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that Sl CYS8 selectively suppresses papain‐like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar‐processing enzyme and serine hydrolase activity. A robust concentration‐dependent inhibition of PLCPs occurred in vitro when purified Sl CYS8 was added to leaf extracts, indicating direct cystatin–PLCP interactions. Activity‐based proteomics revealed that nine different Cathepsin‐L/‐F‐like PLCPs are strongly inhibited by Sl CYS8 in leaves. By contrast, the activity of five other Cathepsin‐B/‐H‐like PLCPs, as well as 87 Ser hydrolases, was unaffected by Sl CYS8. Sl CYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin‐containing proteases from the Resistant‐to‐Desiccation‐21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.

The effect of the unfolded protein response on the production of recombinant proteins in plants.

Recombinant proteins are currently produced through a wide variety of host systems, including yeast, E. coli, insect and mammalian cells. One of the most recent systems developed uses plant cells. While considerable advances have been made in the yields and fidelity of plant-made recombinant proteins, many of these gains have arisen from the development of recombinant factors. This includes elements such as highly effective promoters and untranslated regions, deconstructed viral vectors, silencing inhibitors, and improved DNA delivery techniques. However, unlike other host systems, much of the work on recombinant protein production in plants uses wild-type hosts that have not been modified to facilitate recombinant protein expression. As such, there are still endogenous mechanisms functioning to maintain the health of the cell. The result is that these pathways, such as the unfolded protein response, can actively work to reduce recombinant protein production to maintain the integrity of the cell. This review examines how issues arising from the unfolded protein response have been addressed in other systems, and how these methods may be transferable to plant systems. We further identify several areas of host plant biology that present attractive targets for modification to facilitate recombinant protein production.

Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

Plant‐made pharmaceuticals: Leading products and production platforms

The number of approaches to recombinant protein production in plants is greater than ever before. Development of these new and improved technologies as production platforms for plant-made pharmaceuticals has and will continue to create new commercial opportunities in the pharmaceutical sector. However, it is inevitable that no single system will be optimal for the production of all recombinant proteins of interest in plants due to both the physical characteristics and the envisaged therapeutic application of each product. Here, we review a range of promising product/platform pairs emphasizing synergies during production and in clinical trials.

Production of recombinant proteins in transgenic plants: Practical considerations

This review is based on our recent experience in producing the first commercial recombinant proteins in transgenic plants. We bring forward the issues that have to be considered in the process of selecting and developing a winning transgenic plant production system. From the production point of view, transcription, posttranscription, translation, and posttranslation are important events that can affect the quality and quantity of the final product. Understanding the rules of gene expression is required to develop sound strategies for optimization of recombinant protein production in plants. The level of recombinant protein accumulation is critical, but other factors such as crop selection, handling and processing of transgenic plant material, and downstream processing are equally important when considering commercial production. In some instances, the cost of downstream processing alone may determine the economic viability of a particular plant system. Some of the potential advantages of a plant production system such as the high levels of accumulation of recombinant proteins, glycosylation, compartmentalization within the cell, and natural storage stability in certain organs are incentives for aggressively pursuing recombinant protein production in plants. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 473-484, 1997.

Production of recombinant proteins in transgenic plants: Practical considerations

This review is based on our recent experience in producing the first commercial recombinant proteins in transgenic plants. We bring forward the issues that have to be considered in the process of selecting and developing a winning transgenic plant production system. From the production point of view, transcription, posttranscription, translation, and posttranslation are important events that can affect the quality and quantity of the final product. Understanding the rules of gene expression is required to develop sound strategies for optimization of recombinant protein production in plants. The level of recombinant protein accumulation is critical, but other factors such as crop selection, handling and processing of transgenic plant material, and downstream processing are equally important when considering commercial production. In some instances, the cost of downstream processing alone may determine the economic viability of a particular plant system. Some of the potential advantages of a plant production system such as the high levels of accumulation of recombinant proteins, glycosylation, compartmentalization within the cell, and natural storage stability in certain organs are incentives for aggressively pursuing recombinant protein production in plants. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 473–484, 1997.