Thesis final - after defense-7 - Jacobs University

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Chapter 1 chromatographic method of choice in downstream processing and have been widely used for the purification of proteins, enzymes, antibiotics and vaccines (14, 22, 29). Figure 1: The chromatographic methods based on different protein properties (Adapted from Ref. 20). 1.3. Hydrophobic interaction chromatography (HIC) In industrial biotechnology, the purification of the desired product is very important <strong>after</strong> successful fermentation. There are several chromatography methods available for protein purification based on the physicochemical properties of the proteins. However, HIC can be preferred due to its ability of rapid separation, high resolution and less chances of protein denaturation (23, 24). HIC is based on the reversible interactions between the hydrophobic surface patch of a protein and the hydrophobic surface of a chromatographic medium at moderately high salt concentrations. Proteins contain a variety of amino acids having hydrophilic and hydrophobic residues. Many of the hydrophobic residues are buried in the interior of the proteins, but on exposure to a solvent, a significant proportion of them can appear on the surface. However, the ratio of surface hydrophobicity differs in different 4
Chapter 1 proteins. It is this difference which can be exploited in HIC for protein separations. Protein hydrophobicity is the main physicochemical property which has a decisive role in retention during chromatography. There is no universally agreed single method to calculate protein hydrophobicity, however there is a general agreement that it can be determined by the hydrophobic contribution of the amino acid residues (25, 26). The operating conditions such as mobile phase properties (salt type, ionic strength and pH), stationary phase characteristics (chemical nature of the backbone, type of hydrophobic ligand and substitution level of the resin) and temperature play important roles in HIC (27, 28). Hydrophobic interactions are the most important non covalent forces which have the ability to maintain the native structures of the proteins at high salts concentrations. The basic principle of the HIC is different from size exclusion and ion exchange chromatography (IEC) and thus can be used for the products which have no possibility to be separated by these techniques. HIC is preferred over other chromatographic methods if proteins of similar size or isoelectric point coordinates have to be separated from each other. HIC can also be used as an ideal step, <strong>after</strong> the separation of biomaterials during IEC at high salt concentrations. Due to the entries of several new HIC media and better understanding of the factors affecting the retention, HIC has become one of the most widely used method for purification of proteins such as serum proteins, membrane proteins, nuclear proteins, receptors and recombinant proteins for research and industrial applications (14, 23, 29). The enzymes of commercial importance such as lipases, amylases, streptokinases can be purified utilizing HIC (30). Several expert systems have been produced to exploit the physicochemical properties of the proteins in order to separate the desired product from other contaminants in HIC (31, 32). In most of these approaches, the prediction of protein retention was limited to the use of model proteins instead of the host cell proteome (33). Studies of the host cell proteome which produces the product of interest can help in the design of the process; give in depth knowledge of the target and undesired proteins for the efficient downstream processing. No large scale effort has been made to study the 5
Page 1 and 2: A proteome wide approach to underst
Page 3 and 4: I also want to mention the efforts
Page 5 and 6: Dedication To my father Haji Sher A
Page 7 and 8: Table of Contents 1. Introduction .
Page 9 and 10: 3.3.2.2. Protein distributions on 2
Page 11 and 12: Chapter 1 1. Introduction 1.1. Intr
Page 13: Chapter 1 cell proteome to facilita
Page 17 and 18: Chapter 1 Salt conc. decreasing (Gr
Page 19 and 20: Chapter 1 The ions at the start of
Page 21 and 22: Chapter 1 exposure of hydrophobic r
Page 23 and 24: Chapter 1 based on the surface hydr
Page 25 and 26: Chapter 1 1.5. Analysis of the yeas
Page 27 and 28: Chapter 1 molecular weight impuriti
Page 29 and 30: Chapter 1 followed by its introduct
Page 31 and 32: Chapter 1 protein towards purificat
Page 33 and 34: Chapter 2 EXPERIMENTAL APPROACH
Page 35 and 36: Chapter 2 Yeast cell proteome and c
Page 37 and 38: Chapter 2 ligands (Toyopearl-Hexyl,
Page 39 and 40: Chapter 2 number of spots was count
Page 41 and 42: Chapter 2 (http://www.matrixscience
Page 43 and 44: Chapter 3 RESULTS AND DISCUSSION
Page 45 and 46: Chapter 3 to the retention behavior
Page 47 and 48: Chapter 3 investigate the hydrophob
Page 49 and 50: Chapter 3 the interior of the prote
Page 51 and 52: Chapter 3 A B C Figure 9: The cryst
Page 53 and 54: Chapter 3 of the comparative analys
Page 55 and 56: Chapter 3 Figure 11: Represent the
Page 57 and 58: Chapter 3 Ether, some 2-D gels were
Page 59 and 60: Chapter 3 Figure 13: Represent 2-D
Page 61 and 62: Chapter 3 Table 5. List of the iden
Page 63 and 64: Chapter 3 Table 7. List of the iden
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Chapter 3 amino acids by Cowan-Whit
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Chapter 3 Figure 15 C: Represents t
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Chapter 3 Figure 16 A: Represents t
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Chapter 3 The polarity value for th
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Chapter 3 Figure 17: Represents the
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Chapter 3 published papers with mod
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Chapter 3 ligands revealed less dif
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Chapter 3 3.2. Influence of the dif
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Chapter 3 Table 10: The chemistry o
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Chapter 3 where approximately 50% o
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Chapter 3 3.2.3.2. The electrophore
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Chapter 3 Figure 22: Represent 2-D
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Chapter 3 Table 12. List of the ide
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Chapter 3 Table 14. List of the ide
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Chapter 3 r 2 values from the data
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Chapter 3 Figure 24 C: Represents t
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Chapter 3 approximately 47%. The me
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Chapter 3 Figure 25 B: Represents t
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Chapter 3 values have been reported
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Chapter 3 towards conformational ch
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Chapter 3 among the adsorbents were
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Chapter 3 3.3. Protein distribution
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Chapter 3 3.3.2.2. Protein distribu
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Chapter 3 Figure 29 B: Represent th
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Chapter 3 When the number of smalle
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Chapter 3 and hydrophobic interacti
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Chapter 4 CONCLUSIONS
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Chapter 4 • The base supports rev
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Chapter 4 additional understanding
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References 13. Lienqueo, M.E.; Lese
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References 32. Salgado, J.C.; Rapap
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References 52. B.H.J., H. Accessibl
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References 71. Xindu, G. and Regnie
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References 91. Alonso-Orgaz, S.; Mo
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References 112. Gu, Z. and Glatz, C
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References 132. Reubsaet, J.L.E. an
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Thesis final - after defense-7 - Jacobs University

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