Le virus de l'hépatite C (VHC) est une cause majeure d'affection hépatique chronique, notamment la cirrhose et le cancer du foie. On estime que 170 millions de personnes dans le monde sont des porteurs chroniques du VHC et que 3 à 4 millions de personnes sont infectées chaque année. Un des handicaps majeurs de la recherche sur le VHC est l'absence de systèmes de culture in vitro efficaces et de modèles animaux. Nous avons ainsi choisi une approche recombinante pour l'étude de protéines E1, E2 et p7 du VHC.Les protéines E1, E2 et p7 qui sont impliquées dans des étapes essentielles du cycle viral sont des protéines membranaires. Cependant, l'expression recombinante de cette classe de protéine est extrêmement complexe. En effet, la surexpression des protéines membranaires est souvent toxique pour les cellules hôtes. Ce phénomène est provoqué par l'agrégation ou la dégradation des protéines dans le cytoplasme dû à un manque de membrane disponible pour assurer leur intégration sur la cellule hôte. De plus, la surexpression de protéines membranaires induit la saturation de la machinerie cellulaire liée aux protéines membranaires. Ce détournement empêche le déroulement d'un cycle cellulaire normal et est ainsi fatal pour la cellule hôte. La forte concentration de protéines membranaires ou encore le fait que celles-ci soient hétérologues peut également provoquer la déstabilisation de la membrane de la cellule hôte et de son homéostasie. Afin de nous affranchir de ces limitations, nous avons utilisé une méthode de production des protéines membranaires sous forme native par un système acellulaire en présence de liposomes ; une technologie brevetée par l'université Joseph Fourier et exploitée par la société Synthelis. Dans un premier temps, nous avons procédé à la mise en place du système de production exploitant un lysat bactérien d'E. coli et d'un mélange énergétique complémentaire. Nous avons ensuite utilisé ce system pour étudier la viroporine p7. Cette protéine est essentielle pour la production de particules virales infectieuses et est impliquée dans l'assemblage viral ce qui en fait une cible thérapeutique intéressante. La production de protéoliposomes p7 en grande quantité nous a permis la caractérisation de la protéine par des techniques biochimiques et biophysiques. Nous avons mis en évidence l'inhibition de l'oligomérisation de p7 par le HMA qui ainsi inhibe sa fonction canal ionique. Grâce à la flexibilité du système d'expression acellulaire nous avons caractérisé la structure de la viroporine dans la membrane par réflectivité de neutron et avons confirmé la forme en entonnoir du complexe protéique. Des résultats préliminaires sur les proéoliposomes E1E2 quant à eux permettent d'espérer la production prochaine de particules virales mimant le VHC afin de mieux l'étudier et de lutter contre cette épidémie.L'ensemble de ces résultats confirment la pertinence de l'expression de protéines membranaires sous formes natives en système acellulaire en présence de liposomes. Les protéoliposomes produits constituent des nouveaux outils pour l'étude du VHC et permettent d'envisager de très grandes applications thérapeutiques ainsi que le développement de biomédicaments basés sur l'utilisation de protéines membranaires recombinantes. / The Hepatitis C virus (HCV) is a major cause of chronic liver disease, including cirrhosis and liver cancer. An estimated 170 million people worldwide are chronically infected with HCV and 3 to 4 million people are infected each year. One of the major handicaps of the HCV research is the lack of effective in vitro culture systems and animal models. To adress this issue, we chose a recombinant approach to study the E1, E2 and p7 proteins of HCV.The E1, E2 and p7 proteins are involved in critical steps of the viral cycle. They are membrane proteins, a class of protein that is extremely complex to express. Indeed, overexpression of membrane proteins is often toxic to the host cells. This phenomenon is caused by protein aggregation or degradation in the cytoplasm due to a lack of available membrane space for their integration into the host cell. Moreover, overexpression of membrane proteins induces saturation of the cellular machinery linked to membrane proteins. This diversion prevents the flow of a normal cell cycle and is fatal to the host cell. Destabilization of the host cell's membrane and its homeostatis may also be caused by the high concentration of membrane proteins or their heterologous nature. To circumvent these limitations, we used a method for producing membrane proteins in their native form by a cell-free system in the presence of liposomes; a technology patented by the University Joseph Fourier and licenced by the startup company Synthelis. First, we have set up the cell-free production system using a bacterial lysate from E. coli and a complementary energy mix. We then used this system to study the p7 viroporine. This protein is essential for the production of infectious virus particles and is involved in viral assembly making it an attractive therapeutic target. The production of a large quantity of p7 proteoliposomes allowed us to characterize the protein by biochemical and biophysical techniques. We have demonstrated the inhibition of oligomerization of p7 by HMA, which thereby inhibits its ion channel function. Thanks to the flexibility of the cell-free expression system we have characterized the structure of the viroporine within the membrane in a neutron reflectivity assay and have confirmed the funnel shape of the protein complex. Preliminary results on proteoliposomes E1E2 offer hope for the production viral particles mimicking the hepatitis C virus in order to better study the virus and fight against this epidemic.Together, these results confirm the suitability of the expression of membrane proteins in native forms using a cell-free system in the presence of liposomes. Proteoliposomes products are a new tool for the study of HCV and consideration for very broad therapeutic applications and the development of biopharmaceuticals based on the use of recombinant membrane proteins.

Submitted by Ariane Souza (arianedocarmosouza@gmail.com) on 2018-08-14T02:02:56Z No. of bitstreams: 2 Ariane do Carmo Souza dissertação mestrado.pdf: 1195184 bytes, checksum: e6271bca7a0f0772ac6bb6269483b8fd (MD5) Carta para repositorio Ariane do Carmo Souza.pdf: 105567 bytes, checksum: 2517d898996d34fae9bd02b616df2176 (MD5) / Approved for entry into archive by Alini Demarchi (ri.bar@ufscar.br) on 2018-08-17T12:57:39Z (GMT) No. of bitstreams: 2 Ariane do Carmo Souza dissertação mestrado.pdf: 1195184 bytes, checksum: e6271bca7a0f0772ac6bb6269483b8fd (MD5) Carta para repositorio Ariane do Carmo Souza.pdf: 105567 bytes, checksum: 2517d898996d34fae9bd02b616df2176 (MD5) / Approved for entry into archive by Alini Demarchi (ri.bar@ufscar.br) on 2018-08-17T12:57:52Z (GMT) No. of bitstreams: 2 Ariane do Carmo Souza dissertação mestrado.pdf: 1195184 bytes, checksum: e6271bca7a0f0772ac6bb6269483b8fd (MD5) Carta para repositorio Ariane do Carmo Souza.pdf: 105567 bytes, checksum: 2517d898996d34fae9bd02b616df2176 (MD5) / Made available in DSpace on 2018-09-17T19:25:10Z (GMT). No. of bitstreams: 2 Ariane do Carmo Souza dissertação mestrado.pdf: 1195184 bytes, checksum: e6271bca7a0f0772ac6bb6269483b8fd (MD5) Carta para repositorio Ariane do Carmo Souza.pdf: 105567 bytes, checksum: 2517d898996d34fae9bd02b616df2176 (MD5) Previous issue date: 2018-06-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The alternaria brown spot, caused by Alternaria alternata f sp. citri, causes large economic damages in tangor Murcott (Citrus sinensis Osbeck x Citrus reticulata [L.] Blanco). Its control is carried out through the spraying of agrochemicals, implying up to 15 pulverizations per year, which causes an increase in the production costs of the cultures and damages to the environment. As an alternative, the use of microorganisms, in particular Bacillus spp., has been used to diseases’ control. Therefore, the aim of this work was to evaluate the viability of Bacillus spp in in vitro and in vivo conditions. The methodologies were based on the interactions between biological control agents (Bacillus spp.) and the phytopathogen A. alternata, evaluated by the paired culture technique, by the production of volatile, thermostable and cell-free metabolites by different Bacillus spp. isolates. The molecular identification of the isolates tested and the efficacy of bacterial isolates were evaluated in leaves and plants under greenhouse conditions. The results showed that most of the isolates affected the development of phytopathogen and produced some types of metabolite, being antibiosis one of the probable mechanisms of action of the bacterium. The isolates ACB-01, ACB-07, ACB-08, ACB-18 and ACB-57 presented potential for disease control of A. alternata. / A mancha marrom de alternaria, causada por Alternaria alternata f sp. citri, causa grandes danos econômicos em tangor Murcott (Citrus sinensis L. Osbeck x Citrus reticulata [L.] Blanco). Seu controle é realizado através de pulverizações com agroquímicos, implicando em até 15 pulverizações por ano, o que acarreta em aumento no custo de produção da cultura e prejuízos ao meio ambiente. Como alternativa, o uso de microrganismos, em particular, as bactérias do gênero Bacillus spp., têm sido empregadas para o controle de doenças. Portanto, esse trabalho teve por objetivo avaliar em condições in vitro e in vivo a viabilidade de 47 isolados de Bacillus spp. para o controle da doença. As metodologias foram embasadas nas interações entre agentes de controle biológico (Bacillus spp.) e o fitopatógeno A. alternata avaliadas pela técnica de cultivo pareado, pela produção de metabólitos voláteis, termoestáveis e livre de células por diferentes isolados de Bacillus spp.. Realizou-se, ainda, a identificação molecular dos isolados testados e a eficácia dos isolados da bactéria em folhas destacadas e em plantas, sob condições de casa de vegetação. Os resultados obtidos mostraram que a maioria dos isolados afetou o desenvolvimento do fitopatógeno e produziram algum tipo de metabólito, sendo, a antibiose um dos prováveis mecanismos de ação da bactéria. Os isolados ACB-01, ACB-07, ACB-08, ACB-18 e ACB-57 apresentaram potencial para o biocontrole de A. alternata.

abstract: Protein affinity reagents have aptly gained profound importance as capture reagents and drugs in basic research, biotechnology, diagnostics and therapeutics. However, due to the cost, labor and time associated with production of antibodies focus has recently changed towards potential of peptides to act as protein affinity reagents. Affinity peptides are easy to work with, non-immunogenic, cost effective and amenable to scale up. Even though researchers have developed several affinity peptides, we are far from compiling library of peptides that encompasses entire human proteome. My thesis describes high throughput pipeline that can be used to develop and characterize affinity peptides that bind several discrete sites on target proteins. Chapter 2 describes optimization of cell-free protein expression using commercially available translation systems and well-known leader sequences. Presence of internal ribosome entry site upstream of coding region allows maximal expression in HeLa cell lysate whereas translation enhancing elements are best suited for expression in rabbit reticulocyte lysate and wheat germ extract. Use of optimal vector and cell lysate combination ensures maximum protein expression of DNA libraries. Chapter 3 describes mRNA display selection methodology for developing affinity peptides for target proteins using large diversity DNA libraries. I demonstrate that mild denaturant is not sufficient to increase selection pressure for up to three rounds of selection and increasing number of selection rounds increases probability of finding affinity peptide s. These studies enhance fundamental understanding of mRNA display and pave the way for future optimizations to accelerate convergence of in vitro selections. Chapter 4 describes a high throughput double membrane dot blot system to rapidly screen, identify and characterize affinity peptides obtained from selection output. I used dot blot to screen potential affinity peptides from large diversity of previously ii uncharacterized mRNA display selection output. Further characterization of potential peptides allowed determination of several high affinity peptides from having Kd range 150- 450 nM. Double membrane dot blot is automation amenable, easy and affordable solution for analyzing selection output and characterizing peptides without ne ed for much instrumentation. Together these projects serve as guideline for evolution of cost effective high throughput pipeline for identification and characterization of affinity peptides. / Dissertation/Thesis / Masters Thesis Biochemistry 2014

Perceiving and responding to the environment is key to survival. Using the prokaryotic equivalent of a nervous system – the chemotaxis system – bacteria sense chemical stimuli and respond by adjusting their movement accordingly. In chemotactic bacteria, such as the well-studied E. coli, environmental nutrient sensing is achieved through a membrane embedded protein array that specifically clusters at the cell poles. Signalling to the motor is performed by activation of the CheA kinase, which phosphorylates CheY and CheB. CheY-P tunes the activity of the flagellar motor while CheB-P, together with CheR is involved in adaptation to the stimulus. In E. coli, a dedicated phosphatase terminates the signal. Most bacterial species however, have a much more complex chemotaxis network. Rhodobacter sphaeroides, a model organism for complex chemotaxis systems, has one membrane-embedded chemosensory array and one cytoplasmic chemosensory array, plus several homologs of the E. coli chemotaxis proteins. Signals from both arrays are integrated to control the rotation of a single start-stop flagellar motor. The phosphorelay network has been studied extensively through in vitro phosphotransfer while in vivo studies have established the components of each array and the requirements for formation. Mathematical modelling has also contributed towards inferring connectivities within the signalling network. Starting by constructing a two-hybrid-based interaction network focused on the components of the cytoplasmic chemosensory array, this thesis further addresses its associated adaptation network through a series of in vivo techniques. The swimming behaviour of series of deletion mutants involving the adaptation network of R. sphaeroides is characterised under steady state conditions as well as upon chemotactic stimulation. New connectivities within the R. sphaeroides chemotaxis network are inferred from analysing these data together with results from in vivo photoactivation localisation microscopy of CheB₂. The experimental results are used to propose a new model for chemotaxis in R. sphaeroides.

While protein therapeutics are indispensable in the treatment of a variety of diseases, including cancer, rheumatoid arthritis, and diabetes, key limitations including short half-lives, high immunogenicity, protein instability, and centralized production complicate long-term use and on-demand production. Site-specific polymer conjugation provides a method for mitigating these challenges while minimizing negative impacts on protein activity. However, the location-dependent effects of polymer conjugation are not well understood. Cell-free protein synthesis provides direct access to the synthesis environment and rapid synthesis times, enabling rapid evaluation of multiple conjugation sites on a target protein. Here, work is presented towards developing cell-free protein synthesis as a platform for both design and on-demand production of next generation polymer-protein therapeutics, including (1) eliminating endotoxin contamination in cell-free reagents for simplified therapeutic preparation, (2) improving shelf-stability of cell-free reagents via lyophilization for on-demand production, (3) coupling coarse-grain simulation with high-throughput cell-free protein synthesis to enable rapid identification of optimal polymer conjugation sites, and (4) optimizing cell-free protein synthesis for production of therapeutic proteins

Contexte : Le mélanome est un cancer agressif chez l’homme, développé aux dépens des mélanocytes. L’identification de la mutation BRAF conditionne la prescription d’une thérapie ciblée. L’objectif de ce travail a été de mettre au point dans les tissus tumoraux et dans le sang (cellules tumorales circulantes, ADN libre tumoral plasmatique) des approches technologiques de biologie moléculaire et d’immunohistochimie (IHC) pour identifier des biomarqueurs prédictifs d’une réponse ou de résistance thérapeutique. Nous montrons que l’IHC BRAF (clone VE1, Roche, Ventana) pourrait remplacer l’analyse en biologie moléculaire dans certaines indications, notamment sur un matériel tumoral de petite taille. Parallèlement, nous montrons que la présence de cellules mélanocytaires circulantes [détectées par cytomorphologie (Technique ISET)] chez des patients atteints de mélanome métastatique est un facteur prédictif indépendant de mauvais pronostic de la survie globale. Enfin, nous montrons que le système Biocartis Idylla™ (processus automatisé couplant l’extraction, le séquençage et l’analyse de l’ADN) est sensible et spécifique pour la détection plasmatique des mutations BRAF et NRAS et que cette technique pourrait être indiquée dans le suivi de la maladie résiduelle (apparition de résistance) après traitement des patients atteints de mélanomes métastatiques. Conclusion : L’identification des biomarqueurs tissulaires et sanguins (BRAF, NRAS et CTC) permettent : 1- Une optimisation des délais diagnostiques de la mutation BRAF/NRAS – 2) L’identification de facteurs de mauvais pronostic – 3) De détecter une récidive précoce et de suivre la maladie résiduelle après traitement / Background: Knowledge of the BRAFV600E status is mandatory in metastatic melanoma patients (MMP). Molecular biology is currently the gold standard method for status assessment. The aim of this work was to assess and compare several methods of molecular biology and immunohistochemistry (IHC) in tissue and blood (cell-free circulating tumor DNA, circulating tumor cell (CTC)) to identify predictive biomarkers of response or resistance to targeted treatment. Results: We showed that BRAFV600 IHC could be a substitute for molecular biology in the initial assessment of the BRAFV600E status in MPP. We also found that the presence of circulating tumor cell detetcted by a cytomorphological approach ISET (Isolation by Size of Epithelial Tumor Cell – Rarecells Diagnostics, Paris, France) in MMP is an independent predictor of shorter survival. Then, in a monocentric study conducted at the University of Nice Hospital, we evaluated a novel and fully automated CE-IVD PCR-based system (IdyllaTM, Biocartis, Mechelen, Belgium) for plasmatic BRAF and NRAS mutation detection. We showed that this technology is highly sensitive and specific and provide promising potential to assess tumor progression, identify targets for therapy, and evaluate clinical response to treatment. In conclusion, identification of tissue and blood biomarkers with these technologies allow a quick turnaround-time to BRAF/NRAS diagnosis and improve monitoring of treatment response and development of resistance in metastatic melanoma patients

The purpose of this investigation was to elucidate the changes in cf-DNA as it relates to fluctuations in resistance training workloads and intensities. The relationship between cell free DNA (cf-DNA), C-reactive protein (CRP), creatine kinase (CK), testosterone (T), cortisol (C), testosterone-cortisol ratio (T:C), body mass and body composition were also examined. Eight weightlifters (5 males and 3 females, age = 25 ± 3.5 yr, body mass = 88.3 ± 22.7 kg, height = 173.8 ±8.4 cm) volunteered to participate in this study. Venous blood samples, body mass and body composition were taken six times, each corresponding to the end of a training phase. CK (p = 0.018, η² = 0.409) and CK %Δ (p < 0.001, η² = 0.594) were the only biochemical variables to reach statistical significance at any point. A number of statistically significant correlations were found among variables. VLD4wk was related to CK %Δ (r = 0.86), VLD4wk %Δ was related CK %Δ (r = 0.86) and TID1wk was related to CRP (r = 0.83). cf-DNA %Δ was correlated with CRP and CRP %Δ (r = 0.83 and 0.86, respectively). CRP and CRP %Δ were correlated with BF % (r = 0.94 and 0.92, respectively). CK and CK %Δ were both related to T:C (r = 0.94 and 0.89, respectively) and T:C %Δ (r = 0.87 and 0.86, respectively). The correlation between cf-DNA and CRP suggests that cf-DNA may be a valuable indicator of inflammation in weightlifters.

No description available.

Biological cell populations, such as in tissues or microbial communities, are constantly subject to different sources of noise and variability. Despite this, multicellular systems are still able to function properly because cells coordinate with each other by communication. Using biological model systems to study this multiscalar process can be challenging because of their innate complexity. In this thesis, we address this challenge by building a synthetic multicellular system using bottom-up in vitro assembly approaches. Using this platform, we aim to study the effect of cell-to-cell communication to population variability in a minimal and simplified context. To achieve this, we require a synthetic cell population with (i) quantifiable gene expression dynamics, (ii) customizable population variability, and (iii) intercellular communication. Having these characteristics will allow us to test different initial configurations of population variability and monitor population gene expression dynamics with and without cell-to-cell communication. To generate these synthetic cell populations, reconstituted cell-free expression systems (CFES) are encapsulated into monodisperse-sized liposomes using double-emulsion microfluidics. Both transcription and translation levels are simultaneously monitored and quantified to develop models of cell-free gene expression dynamics and differentiate between bulk and encapsulated formats. Population variability was then incorporated by combining different batches of cells to create distinct subpopulations or by using a two-inlet double-emulsion microfluidic device to generate single populations with a large dispersion of encapsulated DNA template. Lastly, genetic circuits based on the quorum sensing system of Vibrio fischeri are used to implement diffusion-mediated intercellular signalling. Quorum sensing gene circuits in Escherichia coli extract-based CFES were tested in bulk and phase transfer-generated synthetic cells. Together with these experimental systems, corresponding models of synthetic cell populations that can account for population variability and secrete-and-sensing communication are developed using mixed-effects models and moment dynamics. Overall, this work leverages CFES and microfluidic technologies to reproducibly generate a simplified in vitro model of multicellular systems that can be easily monitored spatiotemporally to study multi-scalar processes.:Preface Chapter 1 Bottom-up multicellular systems Chapter 2 Building blocks: cell-free expression and liposomes Chapter 3 Gene expression dynamics in synthetic cell populations Chapter 4 Variability and communication in synthetic cell populations Chapter 5 Modeling variability & communication in synthetic cell populations Summary and outlook Appendices Bibliography / Biologische Zellpopulationen, z.B. in Geweben oder mikrobiellen Gemeinschaften, sind ständig verschiedenen Quellen von Rauschen und Variabilität ausgesetzt. Trotzdem sind multizelluläre Systeme in der Lage, ordnungsgemäß zu funktionieren, weil sich die Zellen durch Kommunikation miteinander abstimmen. Die Verwendung biologischer Modellsysteme zur Untersuchung dieses multiskalaren Prozesses kann aufgrund ihrer angeborenen Komplexität eine Herausforderung darstellen. In dieser Arbeit gehen wir diese Herausforderung an, indem wir ein synthetisches multizelluläres System mit Hilfe von Bottom-up-in vitro-Assembly-Ansätzen aufbauen. Mit Hilfe dieser Plattform wollen wir die Auswirkungen der Kommunikation von Zelle zu Zelle auf die Populationsvariabilität in einem minimalen und vereinfachten Kontext untersuchen. Um dies zu erreichen, benötigen wir eine synthetische Zellpopulation mit (i) quantifizierbarer Genexpressionsdynamik, (ii) anpassbarer Populationsvariabilität und (iii) interzellulärer Kommunikation. Mit diesen Eigenschaften können wir verschiedene Ausgangskonfigurationen der Populationsvariabilität testen und die Genexpressionsdynamik der Population mit und ohne Zell-zu-Zell-Kommunikation beobachten. Um diese synthetischen Zellpopulationen zu erzeugen, werden rekonstituierte zellfreie Expressionssysteme (CFES) mit Hilfe der Doppelemulsions-Mikrofluidik in monodisperse Liposomen eingekapselt. Sowohl die Transkriptions- als auch die Translationsraten werden gleichzeitig überwacht und quantifiziert, um Modelle für die Dynamik der zellfreien Genexpression zu entwickeln und zwischen Bulk- und verkapselten Formaten zu unterscheiden. Die Variabilität der Populationen wurde dann durch die Kombination verschiedener Zellchargen zur Bildung unterschiedlicher Subpopulationen oder durch die Verwendung einer mikrofluidischen Doppelemulsionsvorrichtung mit zwei Einlässen zur Erzeugung einzelner Populationen mit einer großen Streuung der eingekapselten DNA-Vorlage einbezogen. Schließlich werden genetische Schaltkreise auf der Grundlage des Quorum-Sensing-Systems von Vibrio fischeri verwendet, um diffusionsvermittelte interzelluläre Signalübertragung zu implementieren. Quorum-Sensing-Genkreisläufe in CFES auf der Basis von Escherichia coli-Extrakten wurden in synthetischen Zellen getestet, die durch Bulk- und Phasentransfer erzeugt wurden. Zusammen mit diesen experimentellen Systemen wurden entsprechende Modelle synthetischer Zellpopulationen entwickelt, die die Populationsvariabilität und die Sekretions- und Sensing-Kommunikation mit Hilfe von Mixed-Effects-Modellen und Momentendynamik berücksichtigen können. Insgesamt nutzt diese Arbeit CFES- und Mikrofluidik-Technologien, um reproduzierbar ein vereinfachtes in vitro-Modell multizellulärer Systeme zu erzeugen, das leicht raum-zeitlich überwacht werden kann, um multiskalare Prozesse zu untersuchen.:Preface Chapter 1 Bottom-up multicellular systems Chapter 2 Building blocks: cell-free expression and liposomes Chapter 3 Gene expression dynamics in synthetic cell populations Chapter 4 Variability and communication in synthetic cell populations Chapter 5 Modeling variability & communication in synthetic cell populations Summary and outlook Appendices Bibliography

This dissertation reports on the technology of cell-free protein synthesis (CFPS) including 1) stabilized lyophilized cell-free systems and 2) enhanced heterogeneous cell extracts. This work further considers applications of CFPS systems in 1) rapid vaccine development, 2) functional virus-based nanoparticles, 3) site-specific protein immobilization, and 4) expanding the language of biology using unnatural amino acids. CFPS technology is a versatile protein production platform that has many features unavailable in in vivo expression systems. The primary benefit cell-free systems provide is the direct access to the reaction environment, which is no longer hindered by the presence of a cell-wall. The “openness" of the system makes it a compelling candidate for many technologies. One limitation of CFPS is the necessity of freezing for long-term viable storage. We demonstrate that a lyophilized CFPS system is more stable against nonideal storage than traditional CFPS reagents. The Escherichia coli-based CFPS system in this work is limited by the biocatalytic machinery found natively in E. coli. To combat these limitations, exogenous biocatalysts can be expressed during fermentation of cells prepared into extract. We demonstrate that simple adjustments in the fermentation conditions can significantly increase the activity of the heterogeneous extract. Towards virus-based particles and vaccines, we demonstrate that the open nature of CFPS can be utilized for coexpression of virus proteins and self-assembly of virus particles. This technique allows for the rapid production of potential vaccines and novel functional virus-based nanoparticles. Unnatural amino acids expand the effective language of protein biology. Utilizing CFPS as an expression system, we demonstrated that the incorporation of a single specific unnatural amino acid allows for site-specific immobilization, thus stabilizing the protein against elevated temperatures and chemical denaturants. Current unnatural amino acid incorporation technologies are limited to one or few simultaneous incorporations and suffer from low efficiency. This work proposes a system that could potentially allow for upwards of 40 unnatural amino acids to be simultaneously incorporated, effectively tripling the protein code. These projects demonstrate the power and versatility of CFPS technologies while laying the foundation for promising technologies in the field of biotechnology.