Studies In Precipitation Of Proteins From Aqueous Solution

Author : Yu-Chia Cheng
Publisher : ProQuest
ISBN 13 : 9780549388180
Total Pages : pages
Book Rating : 4.3/5 (881 download)

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Download or read book Effects of Salts on the Phase Behavior of Proteins and Protein Mixtures written by Yu-Chia Cheng and published by ProQuest. This book was released on 2008 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.