Imperial College London

Professor Karen Polizzi

Faculty of EngineeringDepartment of Chemical Engineering

Professor of Biotechnology
 
 
 
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Contact

 

+44 (0)20 7594 2851k.polizzi

 
 
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Location

 

411ACE ExtensionSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

97 results found

Kotidis P, Marbiah M, Donini R, Gómez IA, Del Val IJ, Haslam SM, Polizzi KM, Kontoravdi Cet 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.

Journal article

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

Journal article

Aw R, Ashik MR, Islam AAZM, Khan I, Mainuddin M, Islam MA, Ahasan MM, Polizzi KMet al., 2021, Production and purification of an active CRM197 in Pichia pastoris and its immunological characterization using a Vi-typhoid antigen vaccine., Vaccine

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.

Journal article

Stefani I, Blaudin de The F-X, Kontoravdi K, Polizzi Ket al., 2021, Model identifies genetic predisposition of Alzheimer’s disease as key decider in cell susceptibility to stress, International Journal of Molecular Sciences, ISSN: 1422-0067

Journal article

Chee SM, Polizzi K, Freemont P, obled A, Moore S, lai H, morgan R, Thurley Ket al., 2021, A GenoChemetic strategy for derivatization of the violacein natural product scaffold, ACS Chemical Biology, 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.

Journal article

Makrydaki E, Kotidis P, Polizzi KM, Kontoravdi Cet 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

Journal article

Makrydaki E, Marshall O, Heide C, Buldum G, Kontoravdi K, Polizzi Ket 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.

Journal article

Moore S, Tosi T, Bell D, Hleba Y, Freemont P, Polizzi Ket 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.

Journal article

Moore SJ, Hleba YB, Bischoff S, Bell D, Polizzi KM, Freemont PSet 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

Journal article

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.

Journal article

Shmool TA, Martin LK, Bui-Le L, Moya-Ramirez I, Kotidis P, Matthews RP, Venter GA, Kontoravdi C, Polizzi KM, Hallett JPet 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.

Journal article

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.

Journal article

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.

Journal article

Di Blasi R, Marbiah M, Siciliano V, Polizzi K, Ceroni Fet 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.

Journal article

Aw R, De Wachter C, Laukens B, De Rycke R, De Bruyne M, Bell D, Callewaert N, Polizzi KMet 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.

Journal article

Heide C, Buldum G, Moya-Ramirez I, Ces O, Kontoravdi K, Polizzi Ket 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.

Journal article

Shmool TA, Martin LK, Clarke CJ, Bui-Le L, Polizzi KM, Hallett JPet 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

Journal article

Davis B, Backus K, Winter G, Chica R, Li D, Lee SY, He C, Weeks A, Overall C, Hagihara S, Thuronyi B, Kamat S, Chen L-L, Hurtado Guerrero R, Yao S, Mahal LK, Voigt C, Woo C, Strauss E, Kikuchi K, Dore T, Radford S, Li XD, Heo WD, Superti-Furga G, Deans T, Belousov V, Matthews M, Jackson C, Malek S, Waldmann H, Rising A, Jewett M, Stamou D, Parker E, Murakami M, Polizzi K, Hamachi I, Erb T, Joo C, Uesugi M, Prinjha R, Rechavi G, Solano R, Schulman B, David Y, Oslund Ret al., 2021, Voices of chemical biology, Nature Chemical Biology, Vol: 17, Pages: 1-4, ISSN: 1552-4450

Journal article

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.

Journal article

Moya-Ramirez I, Bouton C, Kontoravdi C, Polizzi Ket 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.

Journal article

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.

Journal article

Spice AJ, Aw R, Bracewell DG, Polizzi KMet 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.

Journal article

Gschwend FJ, Hennequin LM, Brandt-Talbot A, Bedoya-Lora F, Kelsall GH, Polizzi K, Fennell PS, Hallett JPet 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.

Journal article

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.

Journal article

Bui-Le L, Clarke CJ, Bröhl A, Brogan APS, Arpino JAJ, Polizzi KM, Hallett Jet 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.

Journal article

Moore SJ, Lai H-E, Kelwick RJR, Chee SM, Bell DJ, Polizzi KM, Freemont PSet al., 2020, Correction to EcoFlex: a multifunctional MoClo kit for E. coli synthetic biology., ACS Synthetic Biology, ISSN: 2161-5063

It has been brought to our attention that the original article contains a typographical error within Figure 1B, part ii. One of the 4-bp overhangs reads “GGAC” and should instead be “GTAC”, as is consistent throughout the original manuscript and deposited AddGene sequences.

Journal article

Riangrungroj P, Polizzi KM, 2020, BeQuIK (Biosensor Engineered Quorum Induced Killing): designer bacteria for destroying recalcitrant biofilms., Microbial Biotechnology, Vol: 13, Pages: 311-314, ISSN: 1751-7915

This opinion piece describes a new design for the remediation of recalcitrant biofilms. It builds on previous work to develop engineered E. coli that recognize quorum sensing signals from pathogens in a biofilm and secrete toxins in response. To solve the challenge of dilute signalling molecules, we propose to use nanobodies and enzymes displayed on the surface of the cells to localize them to the biofilm and degrade the extracellular polymeric substances, thus creating a solution with better 'seek and destroy' capabilities.

Journal article

Spice AJ, Aw R, Bracewell DG, Polizzi KMet al., 2020, Synthesis and assembly of Hepatitis B virus-like particles in a Pichia pastoris cell-free system, Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185

Virus-like particles (VLPs) are supramolecular protein assemblies with the potential for unique and exciting applications in synthetic biology and medicine. Despite the attention VLPs have gained thus far, considerable limitations still persist in their production. Poorly scalable manufacturing technologies and inconsistent product architectures continue to restrict the full potential of VLPs. Cell-free protein synthesis (CFPS) offers an alternative approach to VLP production and has already proven to be successful, albeit using extracts from a limited number of organisms. Using a recently developed Pichia pastoris-based CFPS system, we have demonstrated the production of the model Hepatitis B core antigen VLP as a proof-of-concept. The VLPs produced in the CFPS system were found to have comparable characteristics to those previously produced in vivo and in vitro. Additionally, we have developed a facile and rapid synthesis, assembly and purification methodology that could be applied as a rapid prototyping platform for vaccine development or synthetic biology applications. Overall the CFPS methodology allows far greater throughput, which will expedite the screening of optimal assembly conditions for more robust and stable VLPs. This approach could therefore support the characterization of larger sample sets to improve vaccine development efficiency.

Journal article

Riangrungroj P, Bever CS, Hammock BD, Polizzi KMet al., 2019, A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid insecticide exposure, Scientific Reports, Vol: 9, Pages: 1-9, ISSN: 2045-2322

There is a growing need for low-cost, portable technologies for the detection of threats to the environment and human health. Here we propose a label-free, optical whole-cell Escherichia coli biosensor for the detection of 3-phenoxybenzoic acid (3-PBA), a biomarker for monitoring human exposure to synthetic pyrethroid insecticides. The biosensor functions like a competitive ELISA but uses whole-cells surface displaying an anti-3-PBA VHH as the detection element. When the engineered cells are mixed with 3-PBA-protein conjugate crosslinking that can be visually detected occurs. Free 3-PBA in samples competes with these crosslinks, leading to a detectable change in the output. The assay performance was improved by coloring the cells via expression of the purple-blue amilCP chromoprotein and the VHH expression level was reduced to obtain a limit of detection of 3 ng/mL. The optimized biosensor exhibited robust function in complex sample backgrounds such as synthetic urine and plasma. Furthermore, lyophilization enabled storage of biosensor cells for at least 90 days without loss of functionality. Our whole-cell biosensor is simple and low-cost and therefore has potential to be further developed as a screening tool for monitoring exposure to pyrethroids in low-resource environments.

Journal article

Marques MPC, Boyd AS, Polizzi K, Szita Net al., 2019, Microfluidic devices towards personalized health and wellbeing, JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, Vol: 94, Pages: 2412-2415, ISSN: 0268-2575

Journal article

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