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Author: Weiran Zhang
Publisher:
ISBN:
Category :
Languages : en
Pages : 133
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Book Description
Secretory proteins play critical role in cell survival and pathogenicity and make up approximately 30% of all polypeptides synthesized by gram-negative bacteria. They may be found in a soluble form in the periplasm (the interstitial space between inner and outer membranes), integrated within the outer membrane or excreted outside of the cell in a vesicular or globular form. In all cases, they must first translocate across the inner membrane. A substantial fraction of secretory proteins do so by making use of a cleavable N-terminal extension called signal sequence (or peptide) that is recognized by a dedicated translocation system known as the Sec pathway. Typical Sec-dependent signal sequences have a tripartite structure with a central hydrophobic core and are about 20 residues in length. However, certain secretory proteins implicated in virulence employ Sec-dependent signal peptides that are over 40 residues long. To determine if repetition of canonical, Sec-dependent signal peptides would benefit secretory protein production, we fused the signal sequences of E. carotovora PelB and E. coli OmpA to one another to produce synthetic PelB-OmpA and OmpA-PelB leader peptides. Using periplasmic maltose binding protein (MBP) and outer membrane protein A (OmpA) as model systems, we found that dual signal peptides support Sec-dependent protein translocation and are preferentially cleaved at the signal peptidase I site vicinal to the mature protein. In the case of native OmpA, dual signal peptides increased the requirement for the molecular chaperone Trigger Factor but reduced the accumulation of misfolded precursor and mature species and delayed the acquisition of PBAD promoter mutations that restore cell growth by shutting down recombinant protein synthesis. On the other hand, dual signal sequences did not improve the incorporation of an OmpA1-183-mCherry fusion protein within extracellular outer membrane vesicles (OMVs), as highest yields were obtained with the PelB signal peptide. However, co-expression of periplasmic chaperones (primarily SurA) and components of the outer membrane protein assembly machinery (primarily BamA) improve OMV production by nearly twofold. Overall, our results highlight the potential and limitations of signal sequence and chaperone engineering strategies for the production of secretory proteins and OMVs in E. coli.
Author: Weiran Zhang
Publisher:
ISBN:
Category :
Languages : en
Pages : 133
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Book Description
Secretory proteins play critical role in cell survival and pathogenicity and make up approximately 30% of all polypeptides synthesized by gram-negative bacteria. They may be found in a soluble form in the periplasm (the interstitial space between inner and outer membranes), integrated within the outer membrane or excreted outside of the cell in a vesicular or globular form. In all cases, they must first translocate across the inner membrane. A substantial fraction of secretory proteins do so by making use of a cleavable N-terminal extension called signal sequence (or peptide) that is recognized by a dedicated translocation system known as the Sec pathway. Typical Sec-dependent signal sequences have a tripartite structure with a central hydrophobic core and are about 20 residues in length. However, certain secretory proteins implicated in virulence employ Sec-dependent signal peptides that are over 40 residues long. To determine if repetition of canonical, Sec-dependent signal peptides would benefit secretory protein production, we fused the signal sequences of E. carotovora PelB and E. coli OmpA to one another to produce synthetic PelB-OmpA and OmpA-PelB leader peptides. Using periplasmic maltose binding protein (MBP) and outer membrane protein A (OmpA) as model systems, we found that dual signal peptides support Sec-dependent protein translocation and are preferentially cleaved at the signal peptidase I site vicinal to the mature protein. In the case of native OmpA, dual signal peptides increased the requirement for the molecular chaperone Trigger Factor but reduced the accumulation of misfolded precursor and mature species and delayed the acquisition of PBAD promoter mutations that restore cell growth by shutting down recombinant protein synthesis. On the other hand, dual signal sequences did not improve the incorporation of an OmpA1-183-mCherry fusion protein within extracellular outer membrane vesicles (OMVs), as highest yields were obtained with the PelB signal peptide. However, co-expression of periplasmic chaperones (primarily SurA) and components of the outer membrane protein assembly machinery (primarily BamA) improve OMV production by nearly twofold. Overall, our results highlight the potential and limitations of signal sequence and chaperone engineering strategies for the production of secretory proteins and OMVs in E. coli.
Author: Valeria Mapelli
Publisher: Humana Press
ISBN: 9781493905621
Category : Science
Languages : en
Pages : 0
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Book Description
Yeast Metabolic Engineering: Methods and Protocols provides the widely established basic tools used in yeast metabolic engineering, while describing in deeper detail novel and innovative methods that have valuable potential to improve metabolic engineering strategies in industrial biotechnology applications. Beginning with an extensive section on molecular tools and technology for yeast engineering, this detailed volume is not limited to methods for Saccharomyces cerevisiae, but describes tools and protocols for engineering other yeasts of biotechnological interest, such as Pichia pastoris, Hansenula polymorpha and Zygosaccharomyces bailii. Tools and technologies for the investigation and determination of yeast metabolic features are described in detail as well as metabolic models and their application for yeast metabolic engineering, while a chapter describing patenting and regulations with a special glance at yeast biotechnology closes the volume. Written in the highly successful Methods in Molecular Biology series format, most chapters include an introduction to their respective topic, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and authoritative, Yeast Metabolic Engineering: Methods and Protocols aims to familiarize researchers with the current state of these vital and increasingly useful technologies.
Author: I Barry Holland
Publisher: Elsevier
ISBN: 0080481876
Category : Medical
Languages : en
Pages : 672
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Book Description
ABC Proteins is an in-depth, up-to-date analysis of all that is known about the subject to date. It discusses and compares evolution, biology and mechanism of action of all known ABC proteins, including the first structural studies as well as clinical implications. It will be useful to anyone trying to stay abreast of the latest findings. This book is sure to become a classic and will regularly be updated. - Phylogeny and Evoloution of ABC Transporters - Fundamental Aspects of the Mechanism of Action of ABC Transporters - Prokaryote ABC Transporters - Non-Mammalian Transporters - Multidrug Transporters - ABC Transporters, Physiological Roles and Human Disease - Full color throughout
Author: Christoph Wittmann
Publisher: Springer Science & Business Media
ISBN: 9400745346
Category : Medical
Languages : en
Pages : 391
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Book Description
Systems Metabolic Engineering is changing the way microbial cell factories are designed and optimized for industrial production. Integrating systems biology and biotechnology with new concepts from synthetic biology enables the global analysis and engineering of microorganisms and bioprocesses at super efficiency and versatility otherwise not accessible. Without doubt, systems metabolic engineering is a major driver towards bio-based production of chemicals, materials and fuels from renewables and thus one of the core technologies of global green growth. In this book, Christoph Wittmann and Sang-Yup Lee have assembled the world leaders on systems metabolic engineering and cover the full story – from genomes and networks via discovery and design to industrial implementation practises. This book is a comprehensive resource for students and researchers from academia and industry interested in systems metabolic engineering. It provides us with the fundaments to targeted engineering of microbial cells for sustainable bio-production and stimulates those who are interested to enter this exiting research field.
Author: Yanyan Li
Publisher: Springer
ISBN: 1493910108
Category : Medical
Languages : en
Pages : 113
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Book Description
Lasso peptides form a growing family of fascinating ribosomally-synthesized and post-translationally modified peptides produced by bacteria. They contain 15 to 24 residues and share a unique interlocked topology that involves an N-terminal 7 to 9-residue macrolactam ring where the C-terminal tail is threaded and irreversibly trapped. The ring results from the condensation of the N-terminal amino group with a side-chain carboxylate of a glutamate at position 8 or 9, or an aspartate at position 7, 8 or 9. The trapping of the tail involves bulky amino acids located in the tail below and above the ring and/or disulfide bridges connecting the ring and the tail. Lasso peptides are subdivided into three subtypes depending on the absence (class II) or presence of one (class III) or two (class I) disulfide bridges. The lasso topology results in highly compact structures that give to lasso peptides an extraordinary stability towards both protease degradation and denaturing conditions. Lasso peptides are generally receptor antagonists, enzyme inhibitors and/or antibacterial or antiviral (anti-HIV) agents. The lasso scaffold and the associated biological activities shown by lasso peptides on different key targets make them promising molecules with high therapeutic potential. Their application in drug design has been exemplified by the development of an integrin antagonist based on a lasso peptide scaffold. The biosynthesis machinery of lasso peptides is therefore of high biotechnological interest, especially since such highly compact and stable structures have to date revealed inaccessible by peptide synthesis. Lasso peptides are produced from a linear precursor LasA, which undergoes a maturation process involving several steps, in particular cleavage of the leader peptide and cyclization. The post-translational modifications are ensured by a dedicated enzymatic machinery, which is composed of an ATP-dependent cysteine protease (LasB) and a lactam synthetase (LasC) that form an enzymatic complex called lasso synthetase. Microcin J25, produced by Escherichia coli AY25, is the archetype of lasso peptides and the most extensively studied. To date only around forty lasso peptides have been isolated, but genome mining approaches have revealed that they are widely distributed among Proteobacteria and Actinobacteria, particularly in Streptomyces, making available a rich resource of novel lasso peptides and enzyme machineries towards lasso topologies.
Author: Andreas Kuhn
Publisher: Springer
ISBN: 3030187683
Category : Science
Languages : en
Pages : 501
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Book Description
This book provides an up-to-date overview of the architecture and biosynthesis of bacterial and archaeal cell walls, highlighting the evolution-based similarities in, but also the intriguing differences between the cell walls of Gram-negative bacteria, the Firmicutes and Actinobacteria, and the Archaea. The recent major advances in this field, which have brought to light many new structural and functional details, are presented and discussed. Over the past five years, a number of novel systems, e.g. for lipid, porin and lipopolysaccharide biosynthesis have been described. In addition, new structural achievements with periplasmic chaperones have been made, all of which have revealed amazing details on how bacterial cell walls are synthesized. These findings provide an essential basis for future research, e.g. the development of new antibiotics. The book’s content is the logical continuation of Volume 84 of SCBI (on Prokaryotic Cytoskeletons), and sets the stage for upcoming volumes on Protein Complexes.
Author: A. van Broekhoven
Publisher: Springer Science & Business Media
ISBN: 0306468859
Category : Technology & Engineering
Languages : en
Pages : 436
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Book Description
The present book entitled “Novel Frontiers in the Production of Compounds for Biomedical Uses” can perhaps be placed in its best perspective by the Shakespearean character in The Tempest who exclaimed" What’s past is prologue”. Indeed, this compilation of some of the outstanding presentations in the field of biomedicine made at th the 9 European Congress on Biotechnology (Brussels, Belgium, July 11-15, 1999) not only reflects the achievements of the recent past, but provides a privileged glimpse of the biotechnology that is emerging in the first decade of the new Millennium. It is becoming increasingly apparent that biotechnology is offering biomedicine novel approaches and solutions to develop a sorely needed new generation of biopharmaceuticals. This is all the more necessary because in recent years, new diseases have emerged with extraordinary lethality in all corners of the globe, while age-related chronic illnesses have filled the gap wherever biomedicine has made successful inroads. The rise of antibiotic resistance also poses major threats to public health. Thus, as disease patterns evolve, the rational development of new drugs is becoming urgent, not only for the clinical outcome of patients, but also in optimising the allocation of scarce health care resources through the use of cost-effective productions methods. It is in response to all these challenges that biotechnology offers new strategies that go beyond the more traditional approaches. By the mid-1990’s, the number of recombinant products approved annually for therapeutic use reached double digits. With the advent of the genomics revolution.
Author: Varsha Gupta
Publisher: Springer
ISBN: 9811008752
Category : Technology & Engineering
Languages : en
Pages : 543
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Book Description
This book explores the journey of biotechnology, searching for new avenues and noting the impressive accomplishments to date. It has harmonious blend of facts, applications and new ideas. Fast-paced biotechnologies are broadly applied and are being continuously explored in areas like the environmental, industrial, agricultural and medical sciences. The sequencing of the human genome has opened new therapeutic opportunities and enriched the field of medical biotechnology while analysis of biomolecules using proteomics and microarray technologies along with the simultaneous discovery and development of new modes of detection are paving the way for ever-faster and more reliable diagnostic methods. Life-saving bio-pharmaceuticals are being churned out at an amazing rate, and the unraveling of biological processes has facilitated drug designing and discovery processes. Advances in regenerative medical technologies (stem cell therapy, tissue engineering, and gene therapy) look extremely promising, transcending the limitations of all existing fields and opening new dimensions for characterizing and combating diseases.
Author: Eduardo A. Ceccarelli
Publisher: Frontiers E-books
ISBN: 2889192946
Category : Biotechnology
Languages : en
Pages : 103
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Book Description
With the advent of recombinant DNA technology, expressing heterologous proteins in microorganisms rapidly became the method of choice for their production at laboratory and industrial scale. Bacteria, yeasts and other hosts can be grown to high biomass levels efficiently and inexpensively. Obtaining high yields of recombinant proteins from this material was only feasible thanks to constant research on microbial genetics and physiology that led to novel strains, plasmids and cultivation strategies. Despite the spectacular expansion of the field, there is still much room for progress. Improving the levels of expression and the solubility of a recombinant protein can be quite challenging. Accumulation of the product in the cell can lead to stress responses which affect cell growth. Buildup of insoluble and biologically inactive aggregates (inclusion bodies) lowers the yield of production. This is particularly true for obtaining membrane proteins or high-molecular weight and multi-domain proteins. Also, obtaining eukaryotic proteins in a prokaryotic background (for example, plant or animal proteins in bacteria) results in a product that lack post-translational modifications, often required for functionality. Changing to a eukaryotic host (yeasts or filamentous fungi) may not be a proper solution since the pattern of sugar modifications is different than in higher eukaryotes. Still, many advances in the last couple of decades have provided to researchers a wide variety of strategies to maximize the production of their recombinant protein of choice. Everything starts with the careful selection of the host. Be it bacteria or yeast, a broad list of strains is available for overcoming codon use bias, incorrect disulfide bond formation, protein toxicity and lack of post-translational modifications. Also, a huge catalog of plasmids allows choosing for different fusion partners for improving solubility, protein secretion, chaperone co-expression, antibiotic resistance and promoter strength. Next, controlling culture conditions like temperature, inducer and media composition can bolster recombinant protein production. With this Research Topic, we aim to provide an encyclopedic account of the existing approaches to the expression of recombinant proteins in microorganisms, highlight recent discoveries and analyze the future prospects of this exciting and ever-growing field.
Author: Roger H. Pain
Publisher: Oxford University Press, USA
ISBN: 9780199637881
Category : Science
Languages : en
Pages : 433
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Book Description
Since the publication of the first edition of mechanisms of protein folding in 1994, significant advances in both the technical and conceptual understanding of protein folding. This new edition has been brought up to date in content, context, and authorship and will make the subject accessibleto a wide range of scientists. The emphasis on experimental approaches has benn maintained from the first edition but this time within the explicit context of simulations and energy surfaces. There is an introductory chapter explaining the 'new' model of protein folding, which takes into account theheterogeneity of the starting state. Advances in interpreting observed kinetic data and the development of technology to observe fast folding reactions and characterize intermediate structures have accompanied this new view and are covered in detail. The term 'molten globule'is often usedincorrectly but here the significance of the term is carefully described at different satges of folding. The concept of the transition state, including the complementary approaches of molecular dynamics and protein engineering, is also discussed in detail. In vitro studies provide the molecularbasis for the thermodynamic and kinetic energy minimization of the in vivo processes of protein folding and two of the potentially rate determining reactions are disulphide bond formation and proline isomerization. It has also become increasingly apparent that chaperone proteins play a vital role inprotein folding and other reactions of proteins involoving major conformational change and the molecular details of these processes are discussed in detail in chapter 14. The final chapter describes the centreal importance of protein folding and unfolding reactions in disease and gives claerdefinition of the term 'misfolding'. Studying protein folding in vivo is full of problems and to show how these problems can be overcome in practice, three case studies of three very different types of protein have been included: the small globular protein apomyoglobin; the fibrous protein collagen;and the membrane protein haemagglutinin.
