Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins

Buntru, Matthias; Vogel, Simon; Finnern, Ricarda; Schillberg, Stefan

doi:10.1007/978-1-0716-2241-4_8

Cited by 7 publications

(11 citation statements)

References 19 publications

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“…HBc VLP yields obtained in BYL are comparable to a previously reported E. coli-based CFPS (436 µg/L) (31) and considerably higher than an eukaryotic Pichia pastoris-based CFPS (6.4 µg/mL) (30) and an optimized E. coli cell-based system (3.2 µg/ ml) (18). The BYL was previously reported to reach up to 3mg/ml of a recombinant reporter protein, indicating that further optimizations could be applied to further increment HBc VLP yields (33).…”

Section: Discussionmentioning

confidence: 99%

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system

et al. 2023

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Several vaccine platforms have been developed to fight pathogenic threats, with Virus-Like Particles (VLPs) representing a very promising alternative to traditional platforms. VLPs trigger strong and lasting humoral and cellular immune responses with fewer safety concerns and higher stability than other platforms. The use of extensively characterized carrier VLPs modified with heterologous antigens was proposed to circumvent the viral complexity of specific viruses that could lead to poor VLP assembly and yields. Although carrier VLPs have been successfully produced in a wide variety of cell-based systems, these are limited by low protein yields and protracted clone selection and optimization workflows that limit VLP screening approaches. In response, we have demonstrated the cell-free protein synthesis (CFPS) of several variants of the hepatitis B core (HBc) carrier VLP using a high-yielding tobacco BY-2 lysate (BYL). High VLP yields in the BYL system allowed in-depth characterization of HBc variants. Insertion of heterologous sequences at the spike region of the HBc monomer proved more structurally demanding than at the N-terminus but removal of the C-terminal domain allowed higher particle flexibility and insert acceptance, albeit at the expense of thermal and chemical stability. We also proved the possibility to scale the CFPS reaction up to 1L in batch mode to produce 0.45 grams of the native HBc VLP within a 48-hour reaction window. A maximum yield of 820 µg/ml of assembled VLP particles was observed at the 100µl scale and most remarkably the CFPS reaction was successfully scaled from 50µl to 1L without any reduction in protein yield across this 20,000-fold difference in reaction volumes. We subsequently proved the immunogenicity of BYL-derived VLPs, as flow cytometry and microscopy clearly showed prompt recognition and endocytosis of fluorescently labelled VLPs by human dendritic cells. Triggering of inflammatory cytokine production in human peripheral blood mononuclear cells was also quantitated using a multiplex assay. This research establishes BYL as a tool for rapid production and microscale screening of VLP variants with subsequent manufacturing possibilities across scales, thus accelerating discovery and implementation of new vaccine candidates using carrier VLPs.

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Section: Discussionmentioning

confidence: 99%

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system

et al. 2023

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“…Genes encoding proteins of interest were purchased from gene synthesis providers (Integrated DNA Technologies, Inc.; Twist Bioscience) and transferred into pALiCE01 and pALiCE02 expression vectors (LenioBio GmbH) using the Gibson DNA assembly method 52 . Vectors provided proteins of interest with N- or C-terminal Strep-II tags to enable facile downstream purification, except in the case of adalimumab where tag-less constructs were used.…”

Section: Methodsmentioning

confidence: 99%

“…Generally, CFPS reactions were performed according to the ALiCE® instruction manual and published protocols 52 . Briefly, relevant volumes of BYL product were thawed in a room temperature water bath and template DNA added to final concentration of 5 nM (excepting the heteromeric adalimumab, whereby 5 nM of each heavy and light chain plasmids were used for a total DNA concentration of 10 nM).…”

Section: Methodsmentioning

confidence: 99%

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ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Gupta¹,

Flaskamp²,

Roentgen³

et al. 2022

Preprint

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Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as 'ALiCE', engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 uL microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

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“…The main difficulty encountered when cell-free systems are designed for protein synthesis is the high sensitivity of linear DNA to endonucleases. Numerous solutions to this problem have been investigated, namely, elimination of genes coding for endonucleases from the genome of the cells used for preparing the cell extract, mRNA stabilization, and a decrease in the reaction temperature of the cell-free synthesis in order to lower the activity of residual RNases in the extract [ 106 ].…”

Section: Specific Features Of the Organization Of Nuclear–cytoplasmic...mentioning

confidence: 99%

Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins

Розов

Zagorskaya

Konstantinov

et al. 2022

Plants

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Recombinant proteins are the most important product of current industrial biotechnology. They are indispensable in medicine (for diagnostics and treatment), food and chemical industries, and research. Plant cells combine advantages of the eukaryotic protein production system with simplicity and efficacy of the bacterial one. The use of plants for the production of recombinant proteins is an economically important and promising area that has emerged as an alternative to traditional approaches. This review discusses advantages of plant systems for the expression of recombinant proteins using nuclear, plastid, and mitochondrial genomes. Possibilities, problems, and prospects of modifications of the three parts of the genome in light of obtaining producer plants are examined. Examples of successful use of the nuclear expression platform for production of various biopharmaceuticals, veterinary drugs, and technologically important proteins are described, as are examples of a high yield of recombinant proteins upon modification of the chloroplast genome. Potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated. Although these opportunities have not yet been exploited, potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated.

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Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins

Cited by 7 publications

References 19 publications

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system

ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins

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Plant-Based Cell-Free Transcription and Translation of Recombinant Proteins

Cited by 7 publications

References 19 publications

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system

ALiCE®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins

Contact Info

Product

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About

ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system