Download or read book Production of Complex Heterologous Proteins and Protein Assemblies Using E Coli based Cell free Protein Synthesis written by John Patrick Welsh and published by Stanford University. This book was released on 2011 with total page 195 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Swartz lab has put much effort into understanding the underlying principles of E. coli-based cell-free protein synthesis (CFPS), and the technology has developed into a scalable, affordable platform for producing a wide range of protein targets. Key breakthroughs have included activating central metabolism, stabilization of critical amino acids, controlling the redox environment to produce proteins containing disulfide bonds, and using scale-up technologies to produce proteins at milligram quantities. My work has sought to expand this CFPS technology for producing valuable and complex eukaryotic protein targets by manipulating and optimizing the folding of these proteins in the heterologous CFPS environment. Furthermore, I have sought to apply these advances to specific applications of interest. By modifying a key molecular chaperone native to the eukaryotic endoplasmic reticulum (ER), the Hsp70-family chaperone, BiP, soluble production was increased in CFPS reactions for specific proteins normally secreted through this organelle, namely those from the immunoglobulin superfamily which includes antibodies, T-cell receptors, and many membrane receptors. First, the functional properties of BiP were compared to that of the E. coli Hsp70, DnaK. A fusion protein was then constructed between BiP and the ribosome-binding portion of the E. coli protein, trigger factor, to localize BiP to translating ribosomes. This replicated the native function of BiP, which provides co-translational folding assistance to nascent polypeptides. After verifying its bioactivity, this fusion protein was utilized in CFPS reactions to indicate that the chaperone functions of BiP are specific to proteins normally secreted through the eukaryotic ER, whereas DnaK demonstrates a more general chaperone mechanism. Since the discovery that somatic cells could be reprogrammed back to a pluripotent state through the viral expression of a specific set of transcription factors, there has been great interest in reprogramming using a safer and more clinically relevant protein-based approach. Production of these transcription factor proteins was greatly increased by fusing them to the C-terminus of the solubility partner, IF2 domain 1 (IF2D1). While the fusions provided marginal benefit in molar yields using a CFPS approach, in vivo E. coli expression produced the transcription factors in soluble form. The fusion proteins could be purified in milligram quantities from liter shake-flask cultures, whereas essentially no soluble protein accumulated without the fusion partner. The transcription factor fusions bound specifically to their consensus DNA sequences and partially activated some of their downstream gene targets. Another application utilizing CFPS technology is an enhanced luciferase mutant from the marine copepod, Gaussia princeps (GLuc). GLuc is both the smallest and brightest known luciferase, and previous work from our lab demonstrated that this protein could be produced at higher volumetric yields and specific activities in CFPS compared to conventional protein expression systems. By mutating key residues in the Gaussia luciferase sequence, the luminescence half-life was shown to increase over ten-fold while maintaining the initial specific activity of the wild-type. This improved mutant provides a valuable imaging agent to use in fusions and bioconjugates with other proteins such as those that recognize cell surface markers on cancer cells. In a final application, influenza vaccines were produced using CFPS by isolating specific fragments of the protein hemagglutinin (HA), a viral surface protein. Specific mutations as well as a C-terminal trimerization domain were critical for producing this protein fragment in both its monomeric and native trimeric forms. By using the CFPS platform to incorporate non-natural amino acids (nnAAs) with alkyne functional groups, the HA proteins were covalently 'clicked' to virus-like particles (VLPs) that had surface exposed nnAAs with azide functionality. The VLPs provide an immunogenic delivery platform that efficiently traffics to lymph nodes and allows for co-attachment of other adjuvants in addition to the primary HA antigen. This vaccine platform was characterized and tested in mouse models and compared to both a standard influenza vaccine as well as free HA protein fragments. In summary, CFPS is a valuable and robust method of protein production for a variety of targets. My thesis has sought to use this platform as a means to better understand folding pathways of complex, eukaryotic proteins and improve production of these proteins. To this end, CFPS has been shown to be a valuable method for elucidating and manipulating chaperone function, producing difficult proteins with enhanced function, and as a platform to produce novel vaccines.
Download or read book Production of Complex Heterologous Proteins and Protein Assemblies Using E Coli based Cell free Protein Synthesis written by John Patrick Welsh (#suffix.) and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The Swartz lab has put much effort into understanding the underlying principles of E. coli-based cell-free protein synthesis (CFPS), and the technology has developed into a scalable, affordable platform for producing a wide range of protein targets. Key breakthroughs have included activating central metabolism, stabilization of critical amino acids, controlling the redox environment to produce proteins containing disulfide bonds, and using scale-up technologies to produce proteins at milligram quantities. My work has sought to expand this CFPS technology for producing valuable and complex eukaryotic protein targets by manipulating and optimizing the folding of these proteins in the heterologous CFPS environment. Furthermore, I have sought to apply these advances to specific applications of interest. By modifying a key molecular chaperone native to the eukaryotic endoplasmic reticulum (ER), the Hsp70-family chaperone, BiP, soluble production was increased in CFPS reactions for specific proteins normally secreted through this organelle, namely those from the immunoglobulin superfamily which includes antibodies, T-cell receptors, and many membrane receptors. First, the functional properties of BiP were compared to that of the E. coli Hsp70, DnaK. A fusion protein was then constructed between BiP and the ribosome-binding portion of the E. coli protein, trigger factor, to localize BiP to translating ribosomes. This replicated the native function of BiP, which provides co-translational folding assistance to nascent polypeptides. After verifying its bioactivity, this fusion protein was utilized in CFPS reactions to indicate that the chaperone functions of BiP are specific to proteins normally secreted through the eukaryotic ER, whereas DnaK demonstrates a more general chaperone mechanism. Since the discovery that somatic cells could be reprogrammed back to a pluripotent state through the viral expression of a specific set of transcription factors, there has been great interest in reprogramming using a safer and more clinically relevant protein-based approach. Production of these transcription factor proteins was greatly increased by fusing them to the C-terminus of the solubility partner, IF2 domain 1 (IF2D1). While the fusions provided marginal benefit in molar yields using a CFPS approach, in vivo E. coli expression produced the transcription factors in soluble form. The fusion proteins could be purified in milligram quantities from liter shake-flask cultures, whereas essentially no soluble protein accumulated without the fusion partner. The transcription factor fusions bound specifically to their consensus DNA sequences and partially activated some of their downstream gene targets. Another application utilizing CFPS technology is an enhanced luciferase mutant from the marine copepod, Gaussia princeps (GLuc). GLuc is both the smallest and brightest known luciferase, and previous work from our lab demonstrated that this protein could be produced at higher volumetric yields and specific activities in CFPS compared to conventional protein expression systems. By mutating key residues in the Gaussia luciferase sequence, the luminescence half-life was shown to increase over ten-fold while maintaining the initial specific activity of the wild-type. This improved mutant provides a valuable imaging agent to use in fusions and bioconjugates with other proteins such as those that recognize cell surface markers on cancer cells. In a final application, influenza vaccines were produced using CFPS by isolating specific fragments of the protein hemagglutinin (HA), a viral surface protein. Specific mutations as well as a C-terminal trimerization domain were critical for producing this protein fragment in both its monomeric and native trimeric forms. By using the CFPS platform to incorporate non-natural amino acids (nnAAs) with alkyne functional groups, the HA proteins were covalently 'clicked' to virus-like particles (VLPs) that had surface exposed nnAAs with azide functionality. The VLPs provide an immunogenic delivery platform that efficiently traffics to lymph nodes and allows for co-attachment of other adjuvants in addition to the primary HA antigen. This vaccine platform was characterized and tested in mouse models and compared to both a standard influenza vaccine as well as free HA protein fragments. In summary, CFPS is a valuable and robust method of protein production for a variety of targets. My thesis has sought to use this platform as a means to better understand folding pathways of complex, eukaryotic proteins and improve production of these proteins. To this end, CFPS has been shown to be a valuable method for elucidating and manipulating chaperone function, producing difficult proteins with enhanced function, and as a platform to produce novel vaccines.
Download or read book Cell Free Protein Expression written by James R. Swartz and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 213 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell-free protein synthesis is coming of age! Motivated by an escalating need for efficient protein synthesis and empowered by readily accessible cell-free protein synthesis kits, the technology is expanding both in the range of feasible proteins and in the ways that proteins can be labeled and modified. This volume follows "Cell-Free Translation Systems", edited by Professor Alexander S. Spirin in 2002. Since then, an impressive collection of new work has emerged that demonstrates a substantial expansion of capability. In this volume, we show that proteins now can be efficiently produced using PCR products as DNA templates and that even membrane proteins and proteins with multiple disulfide proteins are obtained at high yields. Many additional advances are also presented. It is an exciting time for protein synthesis technology.
Download or read book Cell free Protein Synthesis of Complex Proteins and Protein Assemblies Containing Post translational Modification written by Aaron Rudy Goerke and published by . This book was released on 2007 with total page 344 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Cell free Protein Synthesis written by Alexander S. Spirin and published by John Wiley & Sons. This book was released on 2014-08-15 with total page 272 pages. Available in PDF, EPUB and Kindle. Book excerpt: With its detailed description of membrane protein expression, high-throughput and genomic-scale expression studies, both on the analytical and the preparative scale, this book covers the latest advances in the field. The step-by-step protocols and practical examples given for each method constitute practical advice for beginners and experts alike.
Download or read book Cell Free Protein Expression written by W. Antoni Kudlicki and published by CRC Press. This book was released on 2007-11-27 with total page 242 pages. Available in PDF, EPUB and Kindle. Book excerpt: Following its inception in the 1950s, cell-free protein synthesis made a tremendous impact on the basic life sciences. The use of cell-free systems was key to understanding molecular mechanisms underlying one of the most complicated processes found in nature: protein translation. Since this time, aggressive cutting-edge research and stiff commerica
Download or read book Recombinant Protein Production with Prokaryotic and Eukaryotic Cells A Comparative View on Host Physiology written by Otto-Wilhelm Merten and published by Springer Science & Business Media. This book was released on 2013-04-17 with total page 396 pages. Available in PDF, EPUB and Kindle. Book excerpt: More then 20 years have passed now since the first recombinant protein producing microorganisms have been developed. In the meanwhile, numerous proteins have been produced in bacteria, yeasts and filamentous fungi, as weIl as higher eukaryotic cells, and even entire plants and animals. Many recombinant proteins are on the market today, and some of them reached substantial market volumes. On the first sight one would expect the technology - including the physiology of the host strains - to be optimised in detail after a 20 year's period of development. However, several constraints have limited the incentive for optimisation, especially in the pharmaceutical industry like the urge to proceed quickly or the requirement to define the production parameters for registration early in the development phase. The additional expenses for registration of a new production strain often prohibits a change to an optimised strain. A continuous optimisation of the entire production process is not feasible for the same reasons.
Download or read book Cell Free Protein Production written by Yaeta Endo and published by Humana. This book was released on 2016-08-23 with total page 261 pages. Available in PDF, EPUB and Kindle. Book excerpt: During the past decade as the data on gene sequences and expression patterns rapidly accumulated, cell-free protein synthesis technology has also experienced a revolution, becoming a powerful tool for the preparation of proteins for their functional and structural analysis. In Cell-Free Protein Production: Methods and Protocols, experts in the field contribute detailed techniques, the uses of which expand deep into the studies of biochemistry, molecular biology, and biotechnology. Beginning briefly with basic methods and historical aspects, the book continues with thorough coverage of protein preparation methods, the preparation of proteins that are generally difficult to prepare in their functional forms, applications of the cell-free technologies to protein engineering, as well as some methods that are expected to constitute a part of future technologies. Written in the highly successful Methods in Molecular BiologyTM series format, the chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Cell-Free Protein Production: Methods and Protocols aims to help researchers continue the growth of the vital exploration of cell-free sciences and technologies in order to better understand the dynamic lives of cells.
Download or read book Cell Free Protein Synthesis written by Manish Biyani and published by BoD – Books on Demand. This book was released on 2012-10-10 with total page 148 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Nobel Prize in Medicine 1968 for interpretation of the genetic code and its function in protein synthesis and in Chemistry 2009 for studies of the structure and function of the ribosome highlighted the ground-breaking experiment performed on May 15, 1961 by Nirenberg and Matthaei and their principal breakthrough on the creation of "cell-free protein synthesis (CFPS) system". Since then the continuous technical advances have revitalized CFPS system as a simple and powerful technology platform for industrial and high-throughput protein production. CFPS yields exceed grams protein per liter reaction volume and offer several advantages including the ability to easily manipulate the reaction components and conditions to favor protein synthesis, decreased sensitivity to product toxicity, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and suitability for miniaturization and high-throughput applications. With these advantages, there is continuous increasing interest in CFPS system among biotechnologists, molecular biologists and medical or pharmacologists.
Download or read book Cell free Macromolecular Synthesis written by Yuan Lu and published by Springer Nature. This book was released on 2023-10-18 with total page 154 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book reviews cell-free production systems, exploring the frontiers in cellular engineering and biotechnology. With contributions from experts in the field, the book offers a comprehensive and up-to-date account of the latest advancements and practical applications. The volume covers a diverse range of topics, beginning with an in-depth analysis of cell-free display techniques for protein evolution, shedding light on the methodologies used to engineer proteins for diverse purposes, followed by an examination of bottom-up synthetic biology employing cell-free protein synthesis. Additionally, it investigates the intricacies of the cell-free synthesis of metalloproteins, elucidating the unique properties and functionalities of these biologically important molecules. In this book, particular attention is given to the integration of cell-free production systems with droplet microfluidics, a pioneering approach that has revolutionized research activities in both academic and industrial settings. Readers will also discover the latest advancements in cell-free protein synthesis and immobilization, and find out more about the eCell technology, which combines cell-free protein synthesis with bio-sensing and remediation, revolutionizing critical areas of study in biotechnology. Together with the companion volume entitled “Cell-free Production: System Development”, both books highlight the research progresses on the basic and applied research of cell-free production systems in the last few years, and are invaluable resources for scholars, researchers, and bioengineers. This book also appeals to enthusiasts of synthetic biology.
Download or read book Cell Free Translation Systems written by A.S. Spirin and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 254 pages. Available in PDF, EPUB and Kindle. Book excerpt: This is a unique book that describes the most recent achievements in the methodology of protein biosynthesis under cell-free conditions. Various versions of cell-free protein-synthesizing systems and their applications to production of individual proteins on a preparative scale are reviewed. The most recent, advanced methodologies, such as continuous-exchange and continuous-flow cell-free systems and novel effecting batch-format cell-free procedures, are considered. Special attention is drawn to the possibilities of structural (NMR; X-ray) analysis of various gene expression products with the use of a new generation of cell-free systems.
Download or read book Kinetic and Constraint based Modeling of E Coli Based Cell free Protein Synthesis written by Nicholas Gabriele Horvath and published by . This book was released on 2019 with total page 179 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell-free protein synthesis is a powerful technology for applications ranging from therapeutics to synthetic biology. Cells are lysed to produce an extract that is used to conduct gene expression in vitro, avoiding the limitations inherent to cell-based systems such as physical barrier of the cell wall and the resource consumption of growth. While useful, this approach has struggled to attain the product yields and reaction times necessary to become a mainstream, industrially viable technology. Metabolic modeling of biological systems can provide insight into underlying mechanism, identify bottlenecks, and suggest system perturbations to improve productivity. Toward this goal, we employed three strategies to model E. coli cell-free protein synthesis: constraint-based, kinetic, and dynamic constraint-based. Sequence-specific constraint-based modeling was used to predict the performance of CFPS for a variety of proteins based on protein length and promoter type. Next an ensemble of kinetic models was used to understand the productivity and yield of E. coli cell-free metabolism under glucose as well as alternative substrates. Model interrogation showed that allosteric control of enzymes was important to system dynamics but not to protein production, and that the most critical pathways for both protein productivity and overall metabolism were oxidative phosphorylation and glycolysis/gluconeogenesis. Dynamic constraint-based modeling highlighted the robustness of protein production to the different pathways of substrate utilization, showed that measurements of central carbon metabolites were most useful to characterize network dynamics, and suggested that despite comprehensive metabolite data, fluxes were still largely unidentifiable. Microfluidic systems, long popular in synthetic biology for their modularity, low cost, and ease of construction, as well as the ability to measure and manipulate metabolites in real-time, have also contributed to improving cell-free protein synthesis. We adapted a glucose oxidase assay to run continuously on a microfluidic device to gain an understanding of in vitro reaction dynamics on-a-chip. An inverse relation was observed between the flow rate through the chip and the extent of reaction. According to a plug-flow reactor model and first-order kinetics, the reaction rate constant was estimated at 46 h-1. Taken together, these modeling approaches and experimental analysis provide an important step toward the goal of point-of-care protein production.
Download or read book Cell Free Synthetic Biology written by Jian Li and published by Frontiers Media SA. This book was released on 2022-01-13 with total page 205 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Advances in Diagnosis and Therapeutic Intervention for Foodborne Parasitic Diseases Volume II written by Wei Cong and published by Frontiers Media SA. This book was released on 2022-06-16 with total page 209 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Escherichia Coli based Cell free Protein Synthesis of Self assembling Particles for Vaccine Production and Gene Therapy written by Noelle Angelica Colant and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Streamlined Extract Preparation for E Coli based Cell free Protein Synthesis and Rapid Site specific Incorporation of Unnatural Amino Acids in Proteins written by Prashanta Shrestha and published by . This book was released on 2012 with total page 83 pages. Available in PDF, EPUB and Kindle. Book excerpt: This work demonstrates simple E. coli extract preparation and improved yield with linear expression templates for further advancements of cell-free protein synthesis system.
Download or read book Cell Free Protein Synthesis written by Kirill Alexandrov and published by Humana. This book was released on 2016-08-27 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell-free protein expression promises to narrow the technological gap between DNA and protein technologies and provide a platform for broad application of synthetic biology principles in the Life Sciences. It is a rapid and high throughput methodology for the conversion of DNA encoded genetic information into protein-mediated biochemical activities. Cell-Free Protein Synthesis: Methods and Protocols brings together the key opinion leaders of cell-free technology development and provides case studies and detailed protocols for the application of cell-free methodology. Chapters cover the main directions in the development of cell-free technologies including several recently developed cell-free systems, as well as a number of applications of cell-free systems ranging from discovery of biofuel enzymes to in vitro assembly of viruses. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Cell-Free Protein Synthesis: Methods and Protocols seeks to serve a wide variety of scientists with its well-honed methodologies.
