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 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 Molecular Biology of the Cell written by and published by . This book was released on 2002 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Protein Synthesis written by Abraham K. Abraham and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 472 pages. Available in PDF, EPUB and Kindle. Book excerpt: During the past decade we have witnessed several major dis coveries in the area of protein synthesis and post-translational modification of protein molecules. In this volume, many of the lat est research developments in these fields are reported by the dis tinguished international group of scientists who presented their state-of-the-art results at the 13th Linderstr0m-Lang Conference held at God0Ysund, Norway, June 14-18, 1983. We feel that the presentation here of so wide a variety of articles on both the molecular and the cellular aspects of protein synthesis will be of considerable value to many scientists working in the area who were unable to attend, as well as to many who are active in related areas. In addition to the research papers, the contents of the six scientific sessions held during the conference have been summarized by the respective session chairmen. These individual summaries provide insightful syntheses of all the recent progress in each field, identify which current problems remain of special inter est, and suggest what the future may hold in the several areas of protein synthesis research covered. Though this volume obviously cannot provide a complete survey of all important ongoing research on the molecular and cellular biology of translational and post-translational events, we are confi dent that it will facilitate a much better understanding of many im portant contemporary problems in research on protein synthesis, including cell differentiation, translational accuracy, protein modifi cation, intracellular transport, and membrane turnover.
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 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 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 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 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 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 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 In Vitro Protein Synthesis of Polyketide Synthase Proteins for Production of Natural Products written by Christopher Antonio Sarmales-Murga and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Heterologous expression of multigene biosynthetic pathways is an essential tool in the study of natural product biosynthesis. Due to its in vivo nature, this process is often limited by, for example, toxicity of the encoded natural product or its biosynthetic proteins, or competition of the biosynthetic proteins with other cellular enzymes for key small molecule building blocks. Cell-free in vitro transcription and translation can overcome some of these limitations. Natural product toxicity is rendered moot in cell-free systems since they are not alive and contain only the necessary proteins, rRNAs, cofactors, substrates, and energy sources for transcription and translation of proteins. As exogenous chemicals can be easily added to the system, building blocks supply issues can be readily solved. We thus investigated using cell-free protein synthesis (CFPS) to biochemically reconstitute the biosynthetic pathway for the fungal polyketide natural product monocillin II. Significant optimization enabled cell-free expression of the full-length monocillin II polyketide synthase (PKS) proteins Rdc5 and Rdc1 directly from plasmids containing their genes under control of the T7 promoter. Correct post-translational modification of the apo-acyl carrier protein domain of the PKS proteins was confirmed by SFP-mediated transfer of a fluorescently modified phosphopantetheinyl group from a chemically modified CoA analog. Unfortunately, treatment of the CFPS produced holo-PKS proteins with their native substrates, malonyl-CoA and NADPH, did not lead to the expected production of detectable levels of monocillin II. Our work suggests that while the CFPS system can generate full length PKS proteins that are sufficiently folded to be recognized, and post-translationally modified by SFP, one or more of the required catalytic domains on these large multidomain proteins is in an inactive state, preventing production of the final product. Identifying non-functional domains, and addressing the issue, may make CFPS an appealing strategy for characterizing PKS biosynthetic gene clusters and prototyping engineered PKS systems.
Download or read book Neuroproteomics written by Oscar Alzate and published by CRC Press. This book was released on 2009-10-26 with total page 356 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this, the post-genomic age, our knowledge of biological systems continues to expand and progress. As the research becomes more focused, so too does the data. Genomic research progresses to proteomics and brings us to a deeper understanding of the behavior and function of protein clusters. And now proteomics gives way to neuroproteomics as we beg
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.
Download or read book The Minimal Cell written by Pier Luigi Luisi and published by Springer Science & Business Media. This book was released on 2010-11-01 with total page 296 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the last ten years there has been a considerable increase of interest on the notion of the minimal cell. With this term we usually mean a cell-like structure containing the minimal and sufficient number of components to be defined as alive, or at least capable of displaying some of the fundamental functions of a living cell. In fact, when we look at extant living cells we realize that thousands of molecules are organized spatially and functionally in order to realize what we call cellular life. This fact elicits the question whether such huge complexity is a necessary condition for life, or a simpler molecular system can also be defined as alive. Obviously, the concept of minimal cell encompasses entire families of cells, from totally synthetic cells, to semi-synthetic ones, to primitive cell models, to simple biomimetic cellular systems. Typically, in the experimental approach to the construction of minimal the main ingredient is the compartment. Lipid vesicles (liposomes) are used to host simple and complex molecular transformations, from single or multiple enzymic reactions, to polymerase chain reactions, to gene expression. Today this research is seen as part of the broader scenario of synthetic biology but it is rooted in origins of life studies, because the construction of a minimal cell might provide biophysical insights into the origins of primitive cells, and the emergence of life on earth. The volume provides an overview of physical, biochemical and functional studies on minimal cells, with emphasis to experimental approaches. 15 International experts report on their innovative contributions to the construction of minimal cells.
Download or read book Cell Free Synthetic Biology written by Seok Hoon Hong and published by MDPI. This book was released on 2020-01-07 with total page 152 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell-free synthetic biology is in the spotlight as a powerful and rapid approach to characterize and engineer natural biological systems. The open nature of cell-free platforms brings an unprecedented level of control and freedom for design compared to in vivo systems. This versatile engineering toolkit is used for debugging biological networks, constructing artificial cells, screening protein library, prototyping genetic circuits, developing new drugs, producing metabolites, and synthesizing complex proteins including therapeutic proteins, toxic proteins, and novel proteins containing non-standard (unnatural) amino acids. The book consists of a series of reviews, protocols, benchmarks, and research articles describing the current development and applications of cell-free synthetic biology in diverse areas.
