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Author: Yaji Tian
Publisher:
ISBN: 9780549072386
Category : Protein engineering
Languages : en
Pages : 104
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Book Description
In addition, E. coli ribose-binding protein (ecRBP) has been stabilized by rational protein engineering to enhance its suitability as a scaffold protein for use in computational design. Several approaches have been exploited to improve the thermostability of ecRBP, including the introduction of mutations to decrease the entropy of the unfolded form, the replacement of un-favored polar amino-acids in the protein core with non-polar residues, the engineering of disulfide bonds, and the incorporation of features from thermophilic RBPs. The stabilizing mutations achieved from these approaches were evaluated individually and then combined in a stepwise manner, resulting in a variant with a melting temperature 17.5°C higher than ecRBP, which can also serve as a stable scaffold protein for biosensor design.
Author: Yaji Tian
Publisher:
ISBN: 9780549072386
Category : Protein engineering
Languages : en
Pages : 104
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Book Description
In addition, E. coli ribose-binding protein (ecRBP) has been stabilized by rational protein engineering to enhance its suitability as a scaffold protein for use in computational design. Several approaches have been exploited to improve the thermostability of ecRBP, including the introduction of mutations to decrease the entropy of the unfolded form, the replacement of un-favored polar amino-acids in the protein core with non-polar residues, the engineering of disulfide bonds, and the incorporation of features from thermophilic RBPs. The stabilizing mutations achieved from these approaches were evaluated individually and then combined in a stepwise manner, resulting in a variant with a melting temperature 17.5°C higher than ecRBP, which can also serve as a stable scaffold protein for biosensor design.
Author: Seymour De Picciotto
Publisher:
ISBN:
Category :
Languages : en
Pages : 136
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Book Description
Investigating protein location and concentration is critical to understanding function. Reagentless biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can detect analytes of interest with high temporal and spatial resolution. However, because these biosensors require laborious empirical screening to develop, their adoption has been limited. Hence, we establish design principles that will facilitate development. In this thesis, we first develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. Following these guidelines, we use the human tenth type III fibronectin domain to engineer new binders against several ligands of the EGFR receptor. Using these binders and others, we design and characterize biosensors based on various target analytes, scaffolds and fluorophores. We observe that analytes can harbor specific binding pockets for the fluorophore, which sharply increase the fluorescence produced upon binding. Furthermore, we demonstrate that a fluorophore conjugated to locally rigid surfaces possesses lower background fluorescence. Based on these newly identified properties, we design biosensors that produce a 100-fold increase in fluorescence upon binding to analyte, about a 10-fold improvement over the previous best biosensor. In order to improve the methodology of reagentless biosensor design, we establish a method for site-specific labeling of proteins displayed on the surface of yeasts. This procedure allows for the screening of libraries of sensors for binding and fluorescence enhancement simultaneously. Finally, we explore an alternative sensor design, based on competitive inhibition of fluorescence quenching.
Author: Huimin Zhao
Publisher: John Wiley & Sons
ISBN: 3527344705
Category : Science
Languages : en
Pages : 41
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Book Description
A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.
Author: Huimin Zhao
Publisher: John Wiley & Sons
ISBN: 3527815112
Category : Science
Languages : en
Pages : 41
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Book Description
A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.
Author: Alfredo Quijano Rubio
Publisher:
ISBN:
Category :
Languages : en
Pages : 107
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Book Description
Traditional protein engineering methods use naturally existing proteins as a starting point and are intrinsically limited to small perturbations of a protein’s original structure and function. Computational de novo protein design is free of such limitations and enables the creation of designer proteins with custom functions and ideal biochemical properties, which make excellent tools to address diverse bioengineering challenges. In this work, I focus on two applications. First, I describe the development of conditionally active cytokine mimics for targeted cancer immunotherapy. We take advantage of the robust folding of a de novo designed Interleukin-2 mimetic protein to design a split version consisting of two components that do not elicit signaling individually but regain activity when co-localized. With this approach, we aim to overcome the severe side-effects of systemic cytokine therapies. Second, I describe the design of modular and tunable biosensors from broadly applicable design principles by using a robust de novo designed protein switch. We demonstrate the modularity of this biosensor platform by designing functional biosensors for multiple analytes of interest.
Author: Genxi Li
Publisher: Elsevier
ISBN: 0128150548
Category : Science
Languages : en
Pages : 380
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Book Description
Nano-inspired Biosensors for Protein Assay with Clinical Applications introduces the latest developments in nano-inspired biosensing, helping readers understand both the fundamentals and frontiers in this rapidly advancing field. In recent decades, there has been increased interest in nano-inspired biosensors for clinical application. Proteins, e.g. antigen-antibody, tumor markers and enzymes are the most important target in disease diagnosis, and a variety of biosensing techniques and strategies have been developed for protein assay. This book brings together all the current literature on the most recent advances of protein analysis and new methodologies in designing new kinds of biosensors for clinical diagnostic use. - Provides a single source of information on the latest developments in the field of biosensors for protein analysis and clinical diagnosis - Focuses on biosensors fabricated with nanomaterials and nanotechnology - Gives detailed methodologies for designing and fabricating nano-inspired biosensors
Author: Jason Davis
Publisher: Royal Society of Chemistry
ISBN: 1847559778
Category : Science
Languages : en
Pages : 270
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Book Description
The interfacing of man-made electronics with redox proteins and enzymes not only tells us a great deal about the levels of sophistication active in biology, but also paves the way to using it in derived sensory devices. Some of these have already had a profound impact on both clinical diagnostics and the quality of life enjoyed by those unfortunate enough to live with disease. Though much remains to be learnt about controlling and optimising these interfacial interactions, their potential uses are, if anything, growing. Written by leaders in the field, this is the only book to focus on the generation of biosensing interfaces with analyses and control at the molecular level. Some of these are enzyme based, others associated with the generation of surfaces for protein-protein recognition. Summaries of state-of-the-art investigations into the interfacing of structurally complex molecular species with electrode surfaces are included along with their design, analysis and potential application. Studies into the "wiring" of biomolecules to man-made surfaces through the use of delocalised "molecular wires" or carbon nanotubes are detailed as are the application of surface chemical and genetic engineering methods to the construction of robust, orientated biomolecular monolayers.
Author: Fred Lisdat
Publisher: Springer
ISBN: 3540752013
Category : Science
Languages : en
Pages : 515
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Book Description
With contributions by numerous experts
Author: Jerome Schultz
Publisher: Springer Science & Business Media
ISBN: 140204058X
Category : Science
Languages : en
Pages : 409
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Book Description
We have come to know that our ability to survive and grow as a nation to a very large degree depends upon our scientific progress. Moreover, it is not enough simply to keep abreast of the rest of the world in scientific matters. 1 We must maintain our leadership. President Harry Truman spoke those words in 1950, in the aftermath of World War II and in the midst of the Cold War. Indeed, the scientific and engineering leadership of the United States and its allies in the twentieth century played key roles in the successful outcomes of both World War II and the Cold War, sparing the world the twin horrors of fascism and totalitarian communism, and fueling the economic prosperity that followed. Today, as the United States and its allies once again find themselves at war, President Truman’s words ring as true as they did a half-century ago. The goal set out in the Truman Administration of maintaining leadership in science has remained the policy of the U.S. Government to this day: Dr. John Marburger, the Director of the Office of Science and Technology (OSTP) in the Executive Office of the President made remarks to that effect during his confirmation hearings in October 2 2001.
Author: Justin Michael Paloni
Publisher:
ISBN:
Category :
Languages : en
Pages : 299
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Book Description
The capability of biosensors to provide highly sensitive and selective molecular detection has enabled development of rapid, inexpensive medical diagnostics. Despite significant advancements in sensor design over the past several decades, most biosensors experience significantly reduced sensitivity in common sensing fluids such as blood and urine. In these mixtures, off-target molecules nonspecifically bind to the sensor surfaces, blocking analyte binding sites and increasing background signal. The self-assembled structure of protein-polymer conjugates presents a potential solution to this issue, offering both biological functionality and a mechanism for excluding many non-analyte molecules in biosensing fluids. Therefore, this thesis explores the use of protein-polymer conjugate thin films as biosensors to minimize nonspecific binding effects during detection in complex mixtures. The first part of this thesis focuses on protein engineering methods to improve the self-assembly of protein-polymer conjugates. It is first demonstrated that oligomerization of low-molecular weight protein blocks significantly enhances ordering quality of the corresponding conjugates. As the degree of oligomerization of the protein block increases, conjugates form ordered phases that display longer-range assembly. Another technique shown to improve protein-polymer conjugate self-assembly is fusion of complementary coiled-coil sequences to the protein block. When proteins bearing these sequences are mixed together in solution, a strongly associative coiled-coil forms, promoting a substantial ordering improvement. Both protein oligomerization and fusion to coiled-coil sequences retain the biological functionality of the protein block, and it is found that protein activity generally scales with conjugate ordering quality. The second part of this thesis explores the capabilities of the polymer block in protein-polymer conjugate thin films to control diffusion into these films. By increasing the molecular weight of this polymer block, larger analyte molecules experience less restricted diffusion into the thin films. Transport studies performed in solutions of the polymer block indicate that most proteins display size-based diffusion following the Stokes-Einstein equation, but some proteins deviate significantly from this behavior due to a combination of protein-protein and protein-polymer interactions. When an analyte molecule is mixed with a protein that diffuses faster than the analyte in these polymer solutions, the sensitivity of the thin film conjugate biosensors towards the analyte is often significantly enhanced. This sensitivity improvement is also observed during detection in mixtures containing the analyte and several proteins, only some of which diffuse faster than the analyte. Accordingly, biosensing measurements using protein-polymer conjugate thin films performed in blood serum and urine solutions, which should contain a variety of proteins that diffuse faster than a given analyte, display a two order of magnitude improvement in sensitivity over traditional surface-based biosensor technologies. Thus, protein-polymer conjugate thin film biosensors can overcome nonspecific binding effects and demonstrate greater sensitivity during measurements performed in complex protein mixtures.
