Download or read book Scale up Optimization and Analysis of Escherichia Coli Extracts for Cell free Protein Synthesis written by James Francis Zawada and published by . This book was released on 2004 with total page 140 pages. Available in PDF, EPUB and Kindle. Book excerpt:
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 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 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 Optimization of Cell free Protein Synthesis by Proteomics and Metabolic Engineering of Escherichia Coli A19 written by Daniel Foshag and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Non Natural Amino Acids written by and published by Academic Press. This book was released on 2009-07-24 with total page 334 pages. Available in PDF, EPUB and Kindle. Book excerpt: By combining the tools of organic chemistry with those of physical biochemistry and cell biology, Non-Natural Amino Acids aims to provide fundamental insights into how proteins work within the context of complex biological systems of biomedical interest. The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. With more than 400 volumes published, each Methods in Enzymology volume presents material that is relevant in today's labs -- truly an essential publication for researchers in all fields of life sciences. - Demonstrates how the tools and principles of chemistry combined with the molecules and processes of living cells can be combined to create molecules with new properties and functions found neither in nature nor in the test tube - Presents new insights into the molecular mechanisms of complex biological and chemical systems that can be gained by studying the structure and function of non-natural molecules - Provides a "one-stop shop" for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods
Download or read book Optimizing Protein Degradation and Improving Energy Regeneration in Escherichia Coli Cell Free Systems and Developing A Simple Method to Dual Site Specifically Label a Protein Using Tryptophan Auxotrophic Escherichia Coli written by Ti Wu and published by . This book was released on 2021 with total page 105 pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation consists of two parts: the first is on optimization and improving protein synthesis and degradation in E. coli cell-free systems, and the second is about a method for incorporating 5-hydroxytryptophan into E. coli release factor 1 protein (RF1) for dual site-specific labeling. Protein synthesis and degradation are fundamental processes in all living cells--the former creates all the protein required for biochemical and cellular functions, and the latter is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. Here we are interested in creating synthetic genetic circuits that function in a cell-free expression system, which will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We further showed that adenosine monophosphate (AMP) accumulates in the cell-free systems, reducing the availability of adenosine nucleotides to energy regeneration. We use class III polyphosphate kinase 2 from Meiothermus ruber (MrPPK2) to mitigate AMP accumulation and improve the performance of protein synthesis and degradation in the cell-free systems. Site-specifically labeling proteins with multiple dyes or molecular moieties is an important yet not-trivial task for many researches, such as when using Föster resonance energy transfer (FRET) to study dynamics of protein conformational change. Many strategies have been devised, but usually done on a case-by-case scenario. Expanded genetic code provided a general platform to incorporate non-canonical amino acids (ncAA), which can also enable multiple site-specific labeling, but it's technically more complicated and not suitable for some specific researches which are in conflict with its technical foundation. We want to develop an easy-to-use protocol for site--specific dual labeling by incorporating 5-hydroxytryptophan using tryptophan auxotroph strain of E. coli, which can provide an extra orthogonal bioconjugation handle additional to conventional amine- or thiol-based labeling method. As demonstration, we incorporated 5-hydroxytryptophan into E. coli release factor 1 (RF1), a protein known to possess two different conformations, and labeled two different fluorophores specifically on 5-hydroxytryptophan and cysteine site. This method provides an easier way to achieve dual- or multi-labeling of protein that will be useful for biochemical or biophysical experiments like FRET, which can help us study the detailed mechanism of RF1-mediated translation termination.
Download or read book Cell free Protein Synthesis written by Alexander S. Spirin and published by John Wiley & Sons. This book was released on 2007-12-03 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 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 Principles and Applications of Cell free Protein Synthesis Systems Scale up written by Alexei Mikhailovich Voloshin and published by . This book was released on 2006 with total page 368 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Cell Free Protein Production written by Yaeta Endo and published by Humana Press. This book was released on 2009-12-04 with total page 0 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 Synthetic Biology written by Christina Smolke and published by John Wiley & Sons. This book was released on 2018-02-28 with total page 532 pages. Available in PDF, EPUB and Kindle. Book excerpt: A review of the interdisciplinary field of synthetic biology, from genome design to spatial engineering. Written by an international panel of experts, Synthetic Biology draws from various areas of research in biology and engineering and explores the current applications to provide an authoritative overview of this burgeoning field. The text reviews the synthesis of DNA and genome engineering and offers a discussion of the parts and devices that control protein expression and activity. The authors include information on the devices that support spatial engineering, RNA switches and explore the early applications of synthetic biology in protein synthesis, generation of pathway libraries, and immunotherapy. Filled with the most recent research, compelling discussions, and unique perspectives, Synthetic Biology offers an important resource for understanding how this new branch of science can improve on applications for industry or biological research.
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 Cell Free Gene Expression written by Ashty S. Karim and published by Humana. This book was released on 2022-01-06 with total page 435 pages. Available in PDF, EPUB and Kindle. Book excerpt: This detailed volume explores perspectives and methods using cell-free expression (CFE) to enable next-generation synthetic biology applications. The first section focuses on tools for CFE systems, including a primer on DNA handling and reproducibility, as well as methods for cell extract preparation from diverse organisms and enabling high-throughput cell-free experimentation. The second section provides an array of applications for CFE systems, such as metabolic engineering, membrane-based and encapsulated CFE, cell-free sensing and detection, and educational kits. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Cell‐Free Gene Expression: Methods and Protocols serves as an ideal guide for researchers seeking technical methods to current aspects of CFE and related applications.
Download or read book Industrial Biotechnology written by Christoph Wittmann and published by John Wiley & Sons. This book was released on 2017-04-10 with total page 789 pages. Available in PDF, EPUB and Kindle. Book excerpt: The latest volume in the Advanced Biotechnology series provides an overview of the main production hosts and platform organisms used today as well as promising future cell factories in a two volume book. Alongside describing tools for genetic and metabolic engineering for strain improvement, the authors also impart topical information on computational tools, safety aspects and industrial-scale production. Following an introduction to general concepts, historical developments and future technologies, the text goes on to cover multi-purpose bacterial cell factories, including those organisms that exploit anaerobic biosynthetic power. Further chapters deal with microbes used for the production of high-value natural compounds and those obtained from alternative raw material sources, concluding with eukaryotic workhorses.
Download or read book Flow Cytometry written by Alice Longobardi Givan and published by John Wiley & Sons. This book was released on 2013-04-10 with total page 309 pages. Available in PDF, EPUB and Kindle. Book excerpt: Flow cytometry continually amazes scientists with its ever-expanding utility. Advances in flow cytometry have opened new directions in theoretical science, clinical diagnosis, and medical practice. The new edition of Flow Cytometry: First Principles provides a thorough update of this now classic text, reflecting innovations in the field while outlining the fundamental elements of instrumentation, sample preparation, and data analysis. Flow Cytometry: First Principles, Second Edition explains the basic principles of flow cytometry, surveying its primary scientific and clinical applications and highlighting state-of-the-art techniques at the frontiers of research. This edition contains extensive revisions of all chapters, including new discussions on fluorochrome and laser options for multicolor analysis, an additionalsection on apoptosis in the chapter on DNA, and new chapters onintracellular protein staining and cell sorting, including high-speed sorting and alternative sorting methods, as well as traditional technology. This essential resource: Assumes no prior knowledge of flow cytometry Progresses with an informal, engaging lecture style from simpleto more complex concepts Offers a clear introduction to new vocabulary, principles of instrumentation, and strategies for data analysis Emphasizes the theory relevant to all flow cytometry, with examples from a variety of clinical and scientific fields Flow Cytometry: First Principles, Second Edition provides scientists, clinicians, technologists, and students with the knowledge necessary for beginning the practice of flow cytometry and for understanding related literature.
Download or read book Recombinant Gene Expression written by Paulina Balbas and published by Springer Science & Business Media. This book was released on 2008-02-04 with total page 505 pages. Available in PDF, EPUB and Kindle. Book excerpt: Since newly created beings are often perceived as either wholly good or bad, the genetic alteration of living cells impacts directly on a symbolic meaning deeply imbedded in every culture. During the earlier years of gene expression research, te- nological applications were confined mainly to academic and industrial laboratories, and were perceived as highly beneficial since molecules that were previously unable to be separated or synthesized became accessible as therapeutic agents. Such were the success stories of hormones, antibodies, and vaccines produced in the bacterium Escherichia coli. Originally this bacterium gained fame among humans for being an unwanted host in the intestine, or worse yet, for being occasionally dangerous and pathogenic. H- ever, it was easily identified in contaminated waters during the 19th century, thus becoming a clear indicator of water pollution by human feces. Tamed, cultivated, and easily maintained in laboratories, its fast growth rate and metabolic capacity to adjust to changing environments fascinated the minds of scientists who studied and modeled such complex phenomena as growth, evolution, genetic exchange, infection, survival, adaptation, and further on—gene expression. Although at the lower end of the complexity scale, this microbe became a very successful model system and a key player in the fantastic revolution kindled by the birth of recombinant DNA technology.
