Author: Thomas C. Evans, Jr. Publisher: Humana Press ISBN: 9781617379680 Category : Medical Languages : en Pages : 310
Book Description
Protein expression in a heterologous host is a cornerstone of biomedical research and of the biotechnology industry. Despite the advanced state of protein expression technology improvements are still needed. For example, membrane proteins constitute a significant percentage of the total cellular proteins but as a class are very difficult to overexpress, especially in a heterologous host. The ideal host would have the ability to express any protein, with relevant post-translational modifications, and be as easy to work with as E. coli. In Heterologous Gene Expression in E. coli: Methods and Protocols, expert scientists intimately familiar with the relevant techniques offer chapters that greatly expand the utility of this expression host. The contributions in this detailed volume describe methods, for example, to successfully express proteins in E. coli that would otherwise form aggregates in this host, to add post-translational modifications, to incorporate non-standard amino acid residues or moieties into E. coli expressed proteins, to identify binding partners, and to express membrane proteins. Written in the highly successful Methods in Molecular BiologyTM format, chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and cutting-edge, Heterologous Gene Expression in E. coli: Methods and Protocols seeks to familiarize the researcher with the myriad of E. coli expression strains available and move E. coli closer to that ideal of the perfect host.
Author: Nicola A. Burgess-Brown Publisher: Humana Press ISBN: 9781493983285 Category : Science Languages : en Pages : 429
Book Description
This detailed volume provides a toolbox for designing constructs, tackling expression and solubility issues, handling membrane proteins and protein complexes, and exploring innovative engineering of E. coli. The topics are largely grouped under four parts: high-throughput cloning, expression screening, and optimization of expression conditions, protein production and solubility enhancement, case studies to produce challenging proteins and specific protein families, as well as applications of E. coli expression. 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, Heterologous Gene Expression in E. coli: Methods and Protocols serves molecular biologists, biochemists and structural biologists, those in the beginning of their research careers to those in their prime, to give both an historical and modern overview of the methods available to express their genes of interest in this exceptional organism.
Author: Eduardo A. Ceccarelli Publisher: Frontiers E-books ISBN: 2889192946 Category : Biotechnology Languages : en Pages : 103
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: Peter E. Vaillancourt Publisher: Springer Science & Business Media ISBN: 1592593011 Category : Science Languages : en Pages : 347
Book Description
Peter E. Vaillancourt presents a collection of popular and emerging methodologies that take advantage of E. coli's ability to quickly and inexpensively express recombinant proteins. The authors focus on two areas of interest: the use of E. coli vectors and strains for production of pure, functional protein, and the use of E. coli as host for the functional screening of large collections of proteins and peptides. Among the cutting-edge techniques demonstrated are those for rapid high-level expression and purification of soluble and functional recombinant protein and those essential to functional genomics, proteomics, and protein engineering.
Author: Paulina Balbas Publisher: Springer Science & Business Media ISBN: 1592597742 Category : Science Languages : en Pages : 505
Book Description
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.
Author: David M. Glover Publisher: Springer ISBN: 1489932461 Category : Medical Languages : en Pages : 231
Book Description
This book was originallyconceived in the form ofa second edition ofa volume published in 1980 in Chapman and Hall's 'OutllneStudies in Biology' series and entitled Genetic Engineering - Cloning DNA. It very rapidly became apparent that with the impact ofrecombinant DNA techniques being feIt in so many areas ofblology, it was going to be difficultifnotimpossible to keepthe bookwithin the space confines of these little monographs. The stays were therefore loosened and the book expanded comfortably to its present size. I hope that this extra space has allowed me to clarify sections ofthe text that were 'heavy going' in the earlierversion. Theextraspace has certainlyallowed me to cover topics that were not mentioned at all in the earlier book. These are primarily to be found in Chapters 7 and 8, which cover the rapid advances that have been recently made in the use ofplantand animal cells as hosts for recombinant DNAmolecules. The develop ment ofother vectors has certainly not stood still over the past four years. This has necessitated a thorough revision ofChapters 3 and 4, which deal with bacteriophage and bacterial plasmid vectors. Numerous techniques for in vitromutagenesis have now been tried and tested allowing me to givecomprehensive coverage ofthisarea in Chapter 2 along with the biochemical techniques used to construct recombinant DNA molecules. Readers with some background knowledge of the approaches to gene cloning will be able to go straight toapart ofthe book in whichthey are specificallyinterested.
Author: Otto-Wilhelm Merten Publisher: Springer Science & Business Media ISBN: 9401597499 Category : Science Languages : en Pages : 396
Book Description
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.
Author: John Patrick Welsh Publisher: Stanford University ISBN: Category : Languages : en Pages : 195
Book Description
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.
Author: Sven-Olof Enfors Publisher: Springer Science & Business Media ISBN: 9783540203117 Category : Medical Languages : en Pages : 264
Book Description
This review series covers trends in modern biotechnology. All aspects of this interdisciplinary technology, where knowledge, methods and expertise are required from chemistry, biochemistry, microbiology, genetics, chemical engineering and computer science, are treated. Electronic version available at http://link.springer.de/series/abe/
Author: Robert E. Glass Publisher: Springer Science & Business Media ISBN: 1468466895 Category : Science Languages : en Pages : 496
Book Description
My aim in writing Gene Function has been to present an up-to-date picture ofthe molecular biology of Escherichia coli. I have not attempted a chronological description, believing that a mechanistic account is more useful for such a highly developed field. I have divided the book into four parts. Part I is a general introduction to bacterial systems, their genetic material, structure, composition and growth. It has seemed desirable to include herein a brief preview of the remaining text, to introduce the nomenclature and to help place subsequent chapters in perspective. The expression of genetic material and its perturbation through mutation is considered in Part II. Part III discusses how the transfer of prokaryotic genetic material can be mediated by plasmids and bacteriophages. It describes the DNA transactions involved (replication, recombination and repair) and ends with a description of the genetic and biochemical techniques employed in the study of gene organisation. Finally, Part IV considers the control of expression of bacterial, plasmid and phage genes. Key reviews are listed at the end of each chapter.
Author: Thomas C. Evans, Jr.
Author: Nicola A. Burgess-Brown
Author: Eduardo A. Ceccarelli
Author: Peter E. Vaillancourt
Author: Paulina Balbas
Author: David M. Glover
Author: Otto-Wilhelm Merten
Author: John Patrick Welsh
Author: Sven-Olof Enfors
Author: Robert E. Glass