101 results found
Marbiah M, Kotidis P, Donini R, et al., 2022, Rapid antibody glycoengineering in Chinese hamster ovary cells., Journal of Visualized Experiments, Vol: 184, Pages: 1-19, ISSN: 1940-087X
Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, t
Makrydaki E, Donini R, Krueger A, et al., 2022, Immobilised enzyme cascade for targeted glycosylation
<jats:title>Abstract</jats:title><jats:p>Glycosylation is a critical post-translational modification of proteins, improving properties such as folding, half-life and functionality. However, glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. Here we describe a platform for <jats:underline>s</jats:underline>eq<jats:underline>u</jats:underline>ential <jats:underline>g</jats:underline>lycosyl<jats:underline>a</jats:underline>tion <jats:underline>r</jats:underline>eactions for <jats:underline>ta</jats:underline>ilo<jats:underline>r</jats:underline>ed su<jats:underline>g</jats:underline>ar s<jats:underline>t</jats:underline>ructures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation <jats:italic>in vitro</jats:italic>. This novel proof-of-concept system is enabled by immobilised enzymes produced with a “one-step immobilisation/purification” method to express, biotinylate <jats:italic>in vivo</jats:italic> and immobilise glycosyltransferases. The immobilised enzymes are used in a reaction cascade mimicking a human-like N-linked glycosylation pathway where promiscuity naturally exists. The enzyme cascade is applied to free glycans, and a monomeric Fc domain expressed in glycoengineered <jats:italic>Pichia pastoris</jats:italic>, yielding near homogeneous glycoforms (>95% conversion). Finally, immobilised β-1,4 galactosyltransferase is used to enhance the galactosylation profile of three different IgGs yielding 80.2 – 96.3 % terminal galactosylation. Enzyme recycling was further demonstrated for 7 cycles, with a combined reaction time greater than 140 hours. The novel SUGAR-TARGET platform is easy to implement, modular and reusable, and therefore can lead to the development of homogeneous glycan structures fo
Moya-Ramirez I, Kotidis P, Marbiah M, et al., 2022, Polymer encapsulation of bacterial biosensors enables co-culture with mammalian cells, ACS Synthetic Biology, Vol: 11, ISSN: 2161-5063
Coexistence of different populations of cells and isolation of tasks can provide enhanced robustness and adaptability or impart new functionalities to a culture. However, generating stable cocultures involving cells with vastly different growth rates can be challenging. To address this, we developed living analytics in a multilayer polymer shell (LAMPS), an encapsulation method that facilitates the coculture of mammalian and bacterial cells. We leverage LAMPS to preprogram a separation of tasks within the coculture: growth and therapeutic protein production by the mammalian cells and l-lactate biosensing by Escherichia coli encapsulated within LAMPS. LAMPS enable the formation of a synthetic bacterial–mammalian cell interaction that enables a living biosensor to be integrated into a biomanufacturing process. Our work serves as a proof-of-concept for further applications in bioprocessing since LAMPS combine the simplicity and flexibility of a bacterial biosensor with a viable method to prevent runaway growth that would disturb mammalian cell physiology.
Spice AJ, Aw R, Polizzi KM, 2022, Cell-Free Protein Synthesis Using Pichia pastoris., Methods Mol Biol, Vol: 2433, Pages: 75-88
Pichia pastoris (syn. Komagataella phaffii) is an industrially relevant recombinant protein platform that has been used to produce over 5000 proteins to date. Cell-free protein synthesis can be used as a screening tool before strain development or for the production of proteins that are difficult or toxic to make in vivo. Here we describe the methods for generating an active cell lysate from P. pastoris using high pressure homogenization and an improved reaction mix which results in high yields of reporter proteins such as luciferase, and complex proteins such as human serum albumin and virus-like particles.
Kotidis P, Marbiah M, Donini R, et al., 2022, Rapid Antibody Glycoengineering in CHO Cells Via RNA Interference and CGE-LIF N-Glycomics., Methods Mol Biol, Vol: 2370, Pages: 147-167
The impact of the glycan distribution on the in vivo function and half-life of monoclonal antibodies has long motivated the genetic engineering of producer cells to achieve structures that enhance efficacy, safety and stability. To facilitate glycoengineering of IgG-producing Chinese hamster ovary cells, we present a rapid protocol that involves the use of RNA interference for the knockdown of genes of interest coupled with capillary gel electrophoresis and laser-induced fluorescence detection (CGE-LIF) for fast, high-throughput glycan analysis. We apply this methodology to the Fut8 gene, responsible for the addition of core fucose, which is a typical target for increasing antibody-dependent cellular cytotoxicity.
Aw R, Ashik MR, Islam AAZM, et al., 2021, Production and purification of an active CRM197 in Pichia pastoris and its immunological characterization using a Vi-typhoid antigen vaccine, Vaccine, Vol: 39, Pages: 7379-7386, ISSN: 0264-410X
CRM197 is a commonly used glycoconjugate carrier that improves the immunogenicity of vaccines, particularly in infants. Despite the advantages of this diphtheria toxoid mutant, low yields, production in inclusion bodies, and the requirement for specific growth conditions have limited the breadth of successful recombinant protein expression platforms available for its expression. We evaluated Pichia pastoris as a production host, using the methanol inducible AOX1 promoter and a modified α-mating factor signal peptide for secretion into the supernatant. Final purified yields >100 mg L−1 culture were achieved when produced in a bioreactor, which is equivalent to the productivity obtained from bioprocesses using the native Corynebacterium diphtheriae host. Recombinant CRM197 was purified to ≥95% homogeneity and showed the expected endonuclease activity. Furthermore, mice immunized with a Salmonella enterica serovar Typhi capsular Vi antigen conjugated to our recombinant CRM197 showed greater than 5-fold increase in immune response. Overall, the results demonstrate that Pichia pastoris is a suitable expression host for the production of high quality CRM197 for vaccine applications.
Lange OJ, Polizzi KM, 2021, Click it or stick it: covalent and non-covalent methods for protein-self assembly, Current Opinion in Systems Biology, Vol: 28, Pages: 100374-100374, ISSN: 2452-3100
Protein complexes are ubiquitous in living systems and have a range of biotechnological applications. However, building protein structures from scratch can be a difficult and laborious process. Here, we review recent developments in protein self-assembly, including a range of tools for covalent and non-covalent assembly of protein structures with user-defined architectures. Key achievements in covalent protein assembly include the development of systems with fast reaction rates and nM affinities. Non-covalent assembly methods have lagged because of the complexity of natural interactions governing protein assembly; but recent developments have created modular methods that are more broadly applicable. On the horizon, we foresee an increasing role for computational protein design tools as key in cementing the role of applications, as opposed to methodology, as the main driving force of research in this field.
Lai H-E, Obled AMC, Chee SM, et al., 2021, GenoChemetic strategy for derivatization of the violacein natural product scaffold, ACS Chemical Biology, Vol: 11, Pages: 2116-2123, ISSN: 1554-8929
Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, new derivatives were generated from 5-brominated violacein analogues via the Suzuki−Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds.
Stefani I, Blaudin de The F-X, Kontoravdi K, et al., 2021, Model identifies genetic predisposition of Alzheimer’s disease as key decider in cell susceptibility to stress, International Journal of Molecular Sciences, Vol: 22, Pages: 1-15, ISSN: 1422-0067
Accumulation of unfolded/misfolded proteins in neuronal cells perturbs endoplasmic reticulum homeostasis, triggering a stress cascade called unfolded protein response (UPR), markers of which are upregulated in Alzheimer’s disease (AD) brain specimens. We measured the UPR dynamic response in three human neuroblastoma cell lines overexpressing the wild-type and two familial AD (FAD)-associated mutant forms of amyloid precursor protein (APP), the Swedish and Swedish-Indiana mutations, using gene expression analysis. The results reveal a differential response to subsequent environmental stress depending on the genetic background, with cells overexpressing the Swedish variant of APP exhibiting the highest global response. We further developed a dynamic mathematical model of the UPR that describes the activation of the three branches of this stress response in response to unfolded protein accumulation. Model-based analysis of the experimental data suggests that the mutant cell lines experienced a higher protein load and subsequent magnitude of transcriptional activation compared to the cells overexpressing wild-type APP, pointing to higher susceptibility of mutation-carrying cells to stress. The model was then used to understand the effect of therapeutic agents salubrinal, lithium, and valproate on signalling through different UPR branches. This study proposes a novel integrated platform to support the development of therapeutics for AD.
Makrydaki E, Kotidis P, Polizzi KM, et al., 2021, Hitting the sweet spot with capillary electrophoresis: advances in N-glycomics and glycoproteomics, CURRENT OPINION IN BIOTECHNOLOGY, Vol: 71, Pages: 182-190, ISSN: 0958-1669
Makrydaki E, Marshall O, Heide C, et al., 2021, Cell-free protein synthesis using Chinese hamster ovary cells, Methods in Enzymology, ISSN: 0076-6879
Cell-Free Protein Synthesis (CFPS) platforms can be used for rapid and flexible expression or proteins. The use CFPS platforms from mammalian, specifically Chinese Hamster Ovary (CHO) cells, offers the possibility of a rapid prototyping platform for recombinant protein production with the capabilities of post-translational modifications (PTMs). In this chapter, we discuss a refined CFPS system based on CHO cells, including: extract preparation, reaction mix composition, and accessory protein supplementation to enhance expression. Specifically, when the CHO cell extract is combined with a truncated version of GADD34 and K3L, stress-induced eIF2 phosphorylation is reduced and inhibition of translation initiation is relieved, increasing yields. A brief summary of the protocol for running the CFPS reactions is also described. Overall, the method is reliable and leads to a highly reproducible expression system. Finally, the advantages and disadvantages of the platform, in addition to expected outcomes, are also discussed.
Moore S, Tosi T, Bell D, et al., 2021, High-yield ‘one-pot’ biosynthesis of raspberry ketone, a high-value fine chemical, Synthetic Biology, Vol: 6, Pages: 1-8, ISSN: 2397-7000
Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction with a yield of ~1-4 mg kg-1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L-1. Herein, we report an efficient cell-free strategy to probe a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one (HBA), into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalysed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a ‘one-pot’ cell-free reaction to produce raspberry ketone at high-yield (61 mg L-1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.
Moore SJ, Hleba YB, Bischoff S, et al., 2021, Refactoring of a synthetic raspberry ketone pathway with EcoFlex (vol 20, 116, 2021), Microbial Cell Factories, Vol: 20, Pages: 1-2, ISSN: 1475-2859
Mastropietro G, Aw R, Polizzi K, 2021, Expression of proteins in Pichia pastoris, Methods in Enzymology, ISSN: 0076-6879
The methylotrophic yeast Pichia pastoris is currently one of the most versatile and popular hosts for the production of heterologous proteins, including industrial enzymes. P. pastoris popularity stems from its ability to grow to high cell densities, producing high titers of secreted heterologous protein with very low amounts of endogenous proteins. Its ability of expressing correctly folded proteins with post-translational modifications make it an excellent candidate for the production of biopharmaceuticals. In addition, production in P. pastoris typically uses the strong, methanol-inducible and tightly regulated promoter (PAOX1), which can result in heterologous protein that constitutes up to 30% of total cell protein upon growth in methanol. In this chapter, we present methodology for the production of secreted recombinant proteins in P. pastoris, and we discuss alternatives to enhance protein production with the desired yield and quality.
Jiménez del Val I, Kyriakopoulos S, Albrecht S, et al., 2021, CHOmpact: a reduced metabolic model of Chinese hamster ovary cells with enhanced interpretability
<jats:title>Abstract</jats:title><jats:p>Metabolic modelling has emerged as a key tool for the characterisation of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimise the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges. Genome scale metabolic models (GeMs) of CHO cells are very large (>6000 reactions) and are, therefore, difficult to constrain to yield physiologically consistent flux distributions. The large scale of GeMs also makes interpretation of their outputs difficult. To address these challenges, we have developed CHOmpact, a reduced metabolic network that encompasses 101 metabolites linked through 144 reactions. Our compact reaction network allows us to deploy multi-objective optimisation and ensure that the computed flux distributions are physiologically consistent. Furthermore, our CHOmpact model delivers enhanced interpretability of simulation results and has allowed us to identify the mechanisms governing shifts in the anaplerotic consumption of asparagine and glutamate as well as an important mechanism of ammonia detoxification within mitochondria. CHOmpact, thus, addresses key challenges of large-scale metabolic models and, with further development, will serve as a platform to develop dynamic metabolic models for the control and optimisation of biopharmaceutical cell culture processes.</jats:p>
Shmool TA, Martin LK, Bui-Le L, et al., 2021, An experimental approach probing the conformational transitions and energy landscape of antibodies: a glimmer of hope for reviving lost therapeutic candidates using ionic liquid, Chemical Science, Vol: 12, Pages: 9528-9545, ISSN: 2041-6520
Understanding protein folding in different environmental conditions is fundamentally important for predicting protein structures and developing innovative antibody formulations. While the thermodynamics and kinetics of folding and unfolding have been extensively studied by computational methods, experimental methods for determining antibody conformational transition pathways are lacking. Motivated to fill this gap, we prepared a series of unique formulations containing a high concentration of a chimeric immunoglobin G4 (IgG4) antibody with different excipients in the presence and absence of the ionic liquid (IL) choline dihydrogen phosphate. We determined the effects of different excipients and IL on protein thermal and structural stability by performing variable temperature circular dichroism and bio-layer interferometry analyses. To further rationalise the observations of conformational changes with temperature, we carried out molecular dynamics simulations on a single antibody binding fragment from IgG4 in the different formulations, at low and high temperatures. We developed a methodology to study the conformational transitions and associated thermodynamics of biomolecules, and we showed IL-induced conformational transitions. We showed that the increased propensity for conformational change was driven by preferential binding of the dihydrogen phosphate anion to the antibody fragment. Finally, we found that a formulation containing IL with sugar, amino acids and surfactant is a promising candidate for stabilising proteins against conformational destabilisation and aggregation. We hope that ultimately, we can help in the quest to understand the molecular basis of the stability of antibodies and protein misfolding phenomena and offer new candidate formulations with the potential to revive lost therapeutic candidates.
Freemont P, 2021, Refactoring of a synthetic raspberry ketone pathway with EcoFlex, Microbial Cell Factories, Vol: 20, Pages: 1-11, ISSN: 1475-2859
Background A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit.Results By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture.Conclusions Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.
Hallett J, 2021, Rhododendron and Japanese Knotweed: invasive species as innovative crops for second generation biofuels, RSC Advances: an international journal to further the chemical sciences, Vol: 11, Pages: 18395-18403, ISSN: 2046-2069
We investigated the potential of two terrestrial biomass invasive species in the United-Kingdom as lignocellulosic biofuel feedstocks: Japanese Knotweed (Fallopia japonica) and Rhododendron (Rhododendron ponticum). We demonstrate that a pretreatment technique using a low-cost protic ionic liquid, the ionoSolv process, can be used for such types of plant species considered as waste, to allow their integration into a biorefinery. N,N,N-Dimethylbutylammonium hydrogen sulfate ([DMBA][HSO4]) was able to fractionate the biomass into a cellulose-rich pulp and a lignin stream at high temperatures (150–170 °C) and short reaction times (15–60 minutes). More than 70–80% of the subsequent cellulose was hydrolysed into fermentable sugars, which were fermented into the renewable energy vector bioethanol.
Di Blasi R, Marbiah M, Siciliano V, et al., 2021, A call for caution in analysing mammalian co-transfection experiments and implications of resource competition in data misinterpretation, Nature Communications, Vol: 12, ISSN: 2041-1723
Transient transfections are routinely used in basic and synthetic biology studies to unravel pathway regulation and to probe and characterise circuit designs. As each experiment has a component of intrinsic variability, reporter gene expression is usually normalized with co-delivered genes that act as transfection controls. Recent reports in mammalian cells highlight how resource competition for gene expression leads to biases in data interpretation, with a direct impact on co-transfection experiments. Here we define the connection between resource competition and transient transfection experiments and discuss possible alternatives. Our aim is to raise awareness within the community and stimulate discussion to include such considerations in future experimental designs, for the development of better transfection controls.
Aw R, De Wachter C, Laukens B, et al., 2021, Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris, Traffic, Vol: 22, Pages: 48-63, ISSN: 1398-9219
The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.
Heide C, Buldum G, Moya-Ramirez I, et al., 2021, Design, development and optimisation of a functional mammalian cell-free protein synthesis platform, Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185
In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS.
Shmool TA, Martin LK, Clarke CJ, et al., 2021, Exploring conformational preferences of proteins: ionic liquid effects on the energy landscape of avidin, CHEMICAL SCIENCE, Vol: 12, Pages: 196-209, ISSN: 2041-6520
Keck FD, Polizzi K, 2021, Microbial interventions are an easier alternative to engineer higher organisms, Microbial Biotechnology, Vol: 14, Pages: 26-30, ISSN: 1751-7907
Advances in synthetic biology have made microbes easier to engineer than ever before. However, synthetic biology in animals and plants has lagged behind. Since it is now known that the phenotype of higher organisms depends largely on their microbiota, we propose that this is an easier route to achieving synthetic biology applications in these organisms.
Moya-Ramirez I, Bouton C, Kontoravdi C, et al., 2020, High resolution biosensor to test the capping level and integrity of mRNAs, Nucleic Acids Research, Vol: 48, Pages: 1-11, ISSN: 0305-1048
5 Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing,translation initiation and stability. Here we describea biosensor designed to detect the presence of capstructures on mRNAs that is also sensitive to mRNAdegradation, so uncapped or degraded mRNAs canbe detected in a single step. The biosensor is basedon a chimeric protein that combines the recognitionand transduction roles in a single molecule. The mainfeature of this sensor is its simplicity, enabling semiquantitative analyses of capping levels with minimalinstrumentation. The biosensor was demonstratedto detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic rangeremained constant with RNAs ranging in size from250 nt to approximately 2700 nt and the biosensorwas able to detect variations in the capping level inincrements of at least 20%, with a limit of detection of2.4 pmol. Remarkably, it also can be applied to morecomplex analytes, such mRNA vaccines and mRNAstranscribed in vivo. This biosensor is an innovativeexample of a technology able to detect analyticallychallenging structures such as mRNA caps. It couldfind application in a variety of scenarios, from qualityanalysis of mRNA-based products such as vaccinesto optimization of in vitro capping reactions.
Aw R, Spice AJ, Polizzi K, 2020, Methods for expression of recombinant proteins using a Pichia pastoris cell-free system, Current protocols in protein science, Vol: 102, ISSN: 1934-3655
Cell‐free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell‐free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell‐free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post‐translational modifications. Here, we describe methods for conducting cell‐free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription‐translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike.
Spice AJ, Aw R, Bracewell DG, et al., 2020, Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort, Synthetic and Systems Biotechnology, Vol: 5, Pages: 137-144, ISSN: 2405-805X
A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a Pichia pastoris cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the P. pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.
Gschwend FJ, Hennequin LM, Brandt-Talbot A, et al., 2020, Towards an environmentally and economically sustainable biorefinery: heavy metal contaminated waste wood as a low-cost feedstock in a low-cost ionic liquid process, Green Chemistry, Vol: 22, Pages: 5032-5041, ISSN: 1463-9262
In the present study, we used a low-cost protic ionic liquid, 1-methylimidazolium chloride, to simultaneously fractionate heavy metal contaminated wood and extract the metals from the wood at elevated temperature and short reaction time. This treatment selectively dissolves the lignin and hemicellulose in the biomass, leaving a solid cellulose-rich pulp, while coordinating and extracting 80–100% of the metal species present in the wood in a one-pot process. The lignin stream was recovered from the liquor and the cellulose was hydrolysed and then fermented into ethanol. The ionic liquid was recycled 6 times and the metals were recovered from the liquor via electrodeposition. This is the first time that highly contaminated waste wood has been integrated into a process which does not produce a contaminated waste stream, but instead valorises the wood as a feedstock for renewable chemicals, materials and fuels, while efficiently recovering the metals, converting a toxic environmental hazard into a rich source of biorenewables. We have therefore used an otherwise problematic waste as a low-cost lignocellulsoic feedstock for a circular bioeconomy concept.
Arpino JAJ, Polizzi KM, 2020, A modular method for directing protein self-assembly, ACS Synthetic Biology, Vol: 9, Pages: 993-1002, ISSN: 2161-5063
Proteins are versatile macromolecules with diverse structure, charge, and function. They are ideal building blocks for biomaterials for drug delivery, biosensing, or tissue engineering applications. Simultaneously, the need to develop green alternatives to chemical processes has led to renewed interest in multienzyme biocatalytic routes to fine, specialty, and commodity chemicals. Therefore, a method to reliably assemble protein complexes using protein-protein interactions would facilitate the rapid production of new materials. Here we show a method for modular assembly of protein materials using a supercharged protein as a scaffolding "hub" onto which target proteins bearing oppositely charged domains have been self-assembled. The physical properties of the material can be tuned through blending and heating and disassembly triggered using changes in pH or salt concentration. The system can be extended to the synthesis of living materials. Our modular method can be used to reliably direct the self-assembly of proteins using small charged tag domains that can be easily encoded in a fusion protein.
Bui-Le L, Clarke CJ, Bröhl A, et al., 2020, Revealing the complexity of ionic liquid-protein interactions through a multi-technique investigation, Communications Chemistry, Vol: 3, ISSN: 2399-3669
Ionic liquids offer exciting possibilities for biocatalysis as solvent properties provide rare opportunities for customizable, energy-efficient bioprocessing. Unfortunately, proteins and enzymes are generally unstable in ionic liquids and several attempts have been made to explain why; however, a comprehensive understanding of the ionic liquid–protein interactions remains elusive. Here, we present an analytical framework (circular dichroism (CD), fluorescence, ultraviolet-visible (UV/Vis) and nuclear magnetic resonance (NMR) spectroscopies, and small-angle X-ray scattering (SAXS)) to probe the interactions, structure, and stability of a model protein (green fluorescent protein (GFP)) in a range (acetate, chloride, triflate) of pyrrolidinium and imidazolium salts. We demonstrate that measuring protein stability requires a similar holistic analytical framework, as opposed to single-technique assessments that provide misleading conclusions. We reveal information on site-specific ionic liquid–protein interactions, revealing that triflate (the least interacting anion) induces a contraction in the protein size that reduces the barrier to unfolding. Robust frameworks such as this are critical to advancing non-aqueous biocatalysis and avoiding pitfalls associated with single-technique investigations.
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