Characterization Of Protein Precipitation Through The Salting Out Effect
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Characterization Of Protein Precipitation Through The Salting Out Effect
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Author : Ying-Chou Shih
language : en
Publisher:
Release Date : 1991
Characterization Of Protein Precipitation Through The Salting Out Effect written by Ying-Chou Shih and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 1991 with categories.
Effects Of Salts On The Phase Behavior Of Proteins And Protein Mixtures
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Author : Yu-Chia Cheng
language : en
Publisher: ProQuest
Release Date : 2008
Effects Of Salts On The Phase Behavior Of Proteins And Protein Mixtures written by Yu-Chia Cheng and has been published by ProQuest this book supported file pdf, txt, epub, kindle and other format this book has been release on 2008 with Precipitation (Chemistry) categories.
Separating proteins in aqueous solutions through fractional precipitation by addition of salts is one of the oldest protein separation methods, and it remains the only purification step for some industrial enzymes and an initial purification step for higher-value therapeutic proteins. However, despite its widespread application, the fundamental characteristics of protein precipitation by salt are still incompletely understood. The primary focus of this thesis is the experimental exploration of the phase behavior of single and binary protein systems during protein precipitation in concentrated ammonium sulfate and sodium chloride solutions, and the effect of solution conditions on protein interactions. Protein precipitation phase behavior of single protein systems was measured as a function of solution conditions for lysozyme and ovalbumin. X-ray powder diffraction (XRD) and optical microscopy were used to characterize the structure of the protein precipitates. These studies showed that crystallization after initial precipitation is feasible even at high ionic strengths if sufficient mixing time is allowed, and such behavior may be a more general phenomenon although different crystallization pathways may be found in some systems and under different conditions; for instance, different pathways are seen in ovalbumin-ammonium sulfate systems as a function of pH. A more important development is the interpretation that emerges of precipitation as a manifestation of a metastable liquid-liquid separation, with the precipitate being a kinetically trapped dense liquid phase. Furthermore, the diversity of possible dense phases (amorphous precipitate, crystal phases or gel) associated with liquid-liquid phase separation shows that understanding the effects of protein interactions of different kinds and at different ranges could have important consequences for the systematic control of protein phase behavior. The phase behavior in selective precipitation of lysozyme-ovalbumin binary mixtures was investigated at various conditions. The best combination of selectivity and recovery of lysozyme was achieved in pH 7 NaCl solutions. At an initial concentration of 30 mg/g H 2 O of lysozyme and 30 mg/g H 2 O of ovalbumin, more than 80% of the lysozyme was collected in precipitate form with 93% purity at ionic strengths above 3 m, while ovalbumin was collected in the supernatant at 80% purity. The purities of both proteins were therefore improved by at least 30% in one step. Moreover, crystallization following initial precipitation was observed in some binary precipitation experiments and indicates that binary precipitation, like single protein precipitation, is a metastable equilibrium state. The observations of selective precipitation behavior were correlated with values of the osmotic second virial coefficient, measured by cross-interaction chromatography (CIC). The results show that interaction measures such as protein self- and cross-interactions can be used as a predictor for general trends and/or inconsistencies in the separation behavior and for optimizing working conditions. Such a mechanistic relationship can obviate the need for extensive trial-and-error methods in order to optimize conditions for protein separations.
Characterization Of Proteins
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Author : Felix Franks
language : en
Publisher: Springer
Release Date : 2007-10-03
Characterization Of Proteins written by Felix Franks and has been published by Springer this book supported file pdf, txt, epub, kindle and other format this book has been release on 2007-10-03 with Science categories.
Proteins are the servants of life. They occur in all component parts of living organisms and are staggering in their functional var- ty, despite their chemical similarity. Even the simplest single-cell organism contains a thousand different proteins, fulfilling a wide range of life-supporting roles. Their production is controlled by the cell’s genetic machinery, and a malfunction of even one protein in the cell will give rise to pathological symptoms. Additions to the total number of known proteins are constantly being made on an increasing scale through the discovery of mutant strains or their production by genetic manipulation; this latter technology has become known as protein engineering. The in vivo functioning of proteins depends critically on the chemical structure of individual peptide chains, but also on the detailed folding of the chains themselves and on their assembly into larger supramolecular structures. The molecules and their fu- tional assemblies possess a limited in vitro stability. Special methods are required for their intact isolation from the source material and for their analysis, both qualitatively and quantitatively. Proteins are also increasingly used as “industrial components,” e.g., in biosensors and immobilized enzymes, because of their specificity, selectivity, and sensitivity. This requires novel and refined proce- ing methods by which the protein isolate can be converted into a form in which it can be utilized.
Protein Precipitation By Electrodialytic Salting Out
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Author : Jeffrey A. Poirier
language : en
Publisher:
Release Date : 1988
Protein Precipitation By Electrodialytic Salting Out written by Jeffrey A. Poirier and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 1988 with Proteins categories.
Affinity Precipitation Of Proteins
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Author : Eva Linné Larsson
language : en
Publisher:
Release Date : 1996
Affinity Precipitation Of Proteins written by Eva Linné Larsson and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 1996 with Precipitation (Chemistry) categories.
Protein Purification
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Author : Robert K. Scopes
language : en
Publisher: Springer Science & Business Media
Release Date : 1993-11-19
Protein Purification written by Robert K. Scopes and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 1993-11-19 with Science categories.
New textbooks at all levels of chemistry appear with great regularity. Some fields such as basic biochemistry, organic re action mechanisms, and chemical thermodynamics are weil represented by many excellent texts, and new or revised editions are published sufficiently often to keep up with progress in research. However, some areas of chemistry, especially many of those taught at the graduate level, suffer from a real lack of up-to-date textbooks. The most serious needs occur in fields that are rapidly changing. Textbooks in these subjects usually have to be written by scientists actually involved in the research that is advancing the field. It is not often easy to persuade such individuals to set time aside to help spread the knowledge they have accumulated. Our goal, in this series, is to pinpoint areas of chemistry where recent progress has outpaced what is covered in any available textbooks, and then seek out and persuade experts in these fields to produce relatively concise but instruc tive introductions to their fields. These should serve the needs of one semester or one quarter graduate courses in chemistry and biochemistry. In so me cases the availability of texts in active research areas should help stimulate the creation of new courses.
The Formation And Structure Of Precipitated Protein Phases
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Author : Daniel G. Greene
language : en
Publisher:
Release Date : 2017
The Formation And Structure Of Precipitated Protein Phases written by Daniel G. Greene and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2017 with categories.
From downstream processing to crystallization screens, protein dense phases, such as gels, aggregates and precipitates, arise during protein solution processing. Several phase separation mechanisms exist that lead to precipitation, including liquidliquid phase separation and crystallization. Competition between these mechanisms and non-equilibrium aggregation mechanisms, such as percolation and gelation, results in complex kinetic phenomena that lead to distinctive microstructures. While the aggregation boundaries have been mapped out for several proteins as a function of various additives, less information is known about the specific microstructures that form within these non-crystalline protein dense phases. Structural biologists have reported qualitative evidence that these dense phases can contain small, highly ordered regions of protein and these data are supported by simulations of colloidal spheres. However, direct experimental measurement of these microstructures is lacking. ☐ First, we utilize small-angle scattering techniques and electron microscopy to measure the microstructure of salted-out ovalbumin. Ovalbumin aggregates into gel beads on the addition of sufficient quantities of ammonium sulfate. Within these gel beads, ovalbumin molecules hierarchically pack into nanocrystallites, approximately 12 nm in size, that assemble into a porous network. Importantly, the results strongly support the hypothesis that non-crystalline protein dense phases can and do contain small, highly ordered regions. ☐ While salted-out ovalbumin exhibits nanocrystals, it is unclear if this structural motif also arises within the dense phases of other proteins or if it is unique to ovalbumin. To address this, the microstructures that form on salting out several other protein species, including lysozyme, ribonuclease A, catalase, and a monoclonal antibody, are then quantified. On shallow quenches nanocrystals ranging in size from 10 to 100 nm develop within these dense phases, further supporting the hypothesis that these macroscopically non-crystalline dense phases do contain highly ordered regions. With recent advances in X-ray sources, we expect that these nanocrystals will be useful for crystallographic measurements. ☐ For deep quenches, a structural motif arises that is consistent with the structure of an arrested spinodal decomposition. The scattering spectra of these deeply quenched samples exhibit a broad peak that is associated with a correlation length scale. The length scale has a power-law dependence on the quench depth and a single power-law index is sufficient to describe the behavior of all the proteins studied, indicating that the behavior may be universal. ☐ The structures discussed above are the result of specific kinetic pathways that arise due to a competition between different growth mechanisms. Therefore, we utilize time-resolved small-angle neutron scattering (TR-SANS) to better understand and characterize these complex pathways. While we are unable to conclusively determine a growth mechanism, TR-SANS reveals that the nanocrystal growth is rapid and can occur in less than an hour. ☐ In many situations, for example during protein purification, precipitation occurs in the presence of multiple protein species. Therefore, we quantify the structure and growth mechanism of a precipitate that forms during the purification of an industrially relevant therapeutic protein when the pH is increased from 3.5 to 5. The precipitate has a mass-fractal microstructure with fractal dimension 2. This structure arises from diffusion-limited aggregation. The process is driven by attractive electrostatic interactions that are modulated by salt and there appears to be a specific ion effect that alters the pH at which the precipitation occurs. ☐ Taken together, these results highlight that precipitation can follow a variety of pathways that lead to very distinct microstructures. A better understanding of these pathways is critical in designing next-generation precipitation processes and the nanocrystals that develop on salting-out may be useful for structural biology. These results establish the first steps in understanding this complex phenomenon on a molecular level.
Amino Acids Peptides And Proteins In Organic Chemistry Analysis And Function Of Amino Acids And Peptides
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Author :
language : en
Publisher: John Wiley & Sons
Release Date : 2013-02-13
Amino Acids Peptides And Proteins In Organic Chemistry Analysis And Function Of Amino Acids And Peptides written by and has been published by John Wiley & Sons this book supported file pdf, txt, epub, kindle and other format this book has been release on 2013-02-13 with Science categories.
This is the last of five books in the Amino Acids, Peptidesand Proteins in Organic Synthesis series. Closing a gap in the literature, this is the only series tocover this important topic in organic and biochemistry. Drawingupon the combined expertise of the international "who's who" inamino acid research, these volumes represent a real benchmark foramino acid chemistry, providing a comprehensive discussion of theoccurrence, uses and applications of amino acids and, by extension,their polymeric forms, peptides and proteins. The practical value of each volume is heightened by theinclusion of experimental procedures. The 5 volumes cover the following topics: Volume 1: Origins and Synthesis of Amino Acids Volume 2: Modified Amino Acids, Organocatalysis and Enzymes Volume 3: Building Blocks, Catalysis and Coupling Chemistry Volume 4: Protection Reactions, Medicinal Chemistry,Combinatorial Synthesis Volume 5: Analysis and Function of Amino Acids and Peptides Volume 5 of this series presents a wealth of methods to analyzeamino acids and peptides. Classical approaches are described, suchas X-ray analysis, chromatographic methods, NMR, AFM, massspectrometry and 2D-gel electrophoresis, as well as newerapproaches, including Surface Plasmon Resonance and arraytechnologies. Originally planned as a six volume series, Amino Acids,Peptides and Proteins in Organic Chemistry now completes withfive volumes but remains comprehensive in both scope andcoverage. ahref="http://eu.wiley.com/WileyCDA/WileyTitle/productCd-3527335463.html"Furtherinformation about the 5 Volume Set and purchasing details can beviewed here./a
Engineering Processes For Bioseparations
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Author : LAURENCE R. WEATHERLEY
language : en
Publisher: Elsevier
Release Date : 2013-10-22
Engineering Processes For Bioseparations written by LAURENCE R. WEATHERLEY and has been published by Elsevier this book supported file pdf, txt, epub, kindle and other format this book has been release on 2013-10-22 with Technology & Engineering categories.
The use of biotechnology in chemical synthesis offers up numerous advantages to the engineer in the process industries, but it also presents a number of fundamental challenges and difficulties which impinge directly on separation process requirements. The use of biochemical separations has grown significantly during the past decade, and is especially used in process industries such as healthcare and food processing. However it is becoming increasingly more important in areas such as recycling and waste-water treatment and as industry shifts towards cleaner processes biochemical separations will continue to grow. The two main objectives of this book are to focus on the application of existing separation process techniques to the recovery and purification of biologically derived products and to examine the state of knowledge of new techniques which have future potential. Within these objectives the complexities and breadth of problems associated with biological separations are discussed, specific engineering techniques are featured and their adaptation to biochemical separations are highlighted.
Principles And Reactions Of Protein Extraction Purification And Characterization
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Author : Hafiz Ahmed
language : en
Publisher: CRC Press
Release Date : 2017-07-27
Principles And Reactions Of Protein Extraction Purification And Characterization written by Hafiz Ahmed and has been published by CRC Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2017-07-27 with Medical categories.
Principles and Reactions of Protein Extraction, Purification, and Characterization provides the mechanisms and experimental procedures for classic to cutting-edge techniques used in protein extraction, purification, and characterization. The author presents the principles and reactions behind each procedure and uses tables to compare the different