Thesis final - after defense-7 - Jacobs University

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Chapter 3 The correlation coefficients (r 2 ) of ASH with retention of proteins were 0.93, 0.83 and 0.96 for Toyopearl-Phenyl, Sepharose-Phenyl and Source-Phenyl, respectively. The correlation coefficient reported in the Sepharose-Phenyl case was less than Toyopearl-Phenyl and Source-Phenyl; the reason could be the heterogeneous distribution of the hydrophobic hotspots on the base support. This kind of behavior was previously reported with chromatographic separation of model proteins by Sepharose-Phenyl, where proteins of similar ASH have exhibited different retention times (63). In the previous reports, model proteins have been used to correlate ASH with the retention behavior. This correlation was never examined as a function of the complex cell proteome (26, 27). The correlation of ASH with retention behavior was explored for the first time in this work with the yeast cell proteome. The experimental data about salt concentrations and corresponding hydrophobicity ranges at different elution stages can be exploited to predict the separation of recombinant proteins employing yeast as a host cell proteome. The separation of recombinant proteins by HIC can also be predicted in other host cell proteomes, once its ASH is calculated. ASH was the only property which has revealed the trend lines to partially differentiate among the base supports. 3.2.3.3.4. Other protein properties affecting the chromatographic behavior The average flexibility was calculated for the tabulated proteins using the scale proposed by Bhaskaran and Ponnuswamy (Tables 12-14) (98). The average flexibility has revealed a statistically significant (r 2 = 0.80; p < 0.0001) direct correlation with the protein retention during chromatography (Figure 27). The average flexibility has been reported before for its direct relationship with the retention time of model proteins in HIC (62). This could be due to the presence of cystein residues in a protein forming disulfide bridges. A protein with more cystein content / and or disulfide bridges will be highly stable and less flexible towards conformational changes. In contrast, proteins with less cystein residues will be highly flexible 95
Chapter 3 towards conformational changes and when it comes in contact of HIC surface, it will unfold or expose the internal surface in order to increase hydrophobic interactions and result in longer retention during chromatography (33, 62, 128). Figure 27: Represents the average flexibility calculated by Bhaskaran and Ponnuswamy’s scale. The protein average flexibility values of the three base supports were correlated with the respective salt concentrations where the fractions were collected. The error bars represent the standard deviations of the average flexibility values in each fraction. The statistical analysis revealed a significant correlation (r 2 = 0.80; p < 0.0001). It was also reported by Tanford that proteins with native conformations were completely inflexible and conformational entropy was zero. In contrast, the unfolded proteins have high flexibility towards conformational changes (25). This revealed that proteins with less flexibility had less conformational entropy and high stability. In this work, average flexibility was correlated with the retention of proteins derived from a host cell proteome. Some proteins 96
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A proteome wide approach to underst
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I also want to mention the efforts
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Dedication To my father Haji Sher A
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Table of Contents 1. Introduction .
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3.3.2.2. Protein distributions on 2
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Chapter 1 1. Introduction 1.1. Intr
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Chapter 1 cell proteome to facilita
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Chapter 1 proteins. It is this diff
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Chapter 1 Salt conc. decreasing (Gr
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Chapter 1 The ions at the start of
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Chapter 1 exposure of hydrophobic r
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Chapter 1 based on the surface hydr
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Chapter 1 1.5. Analysis of the yeas
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Chapter 1 molecular weight impuriti
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Chapter 1 followed by its introduct
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Chapter 1 protein towards purificat
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Chapter 2 EXPERIMENTAL APPROACH
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Chapter 2 Yeast cell proteome and c
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Chapter 2 ligands (Toyopearl-Hexyl,
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Chapter 2 number of spots was count
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Chapter 2 (http://www.matrixscience
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Chapter 3 RESULTS AND DISCUSSION
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Chapter 3 to the retention behavior
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Chapter 3 investigate the hydrophob
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Chapter 3 the interior of the prote
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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
Page 65 and 66: Chapter 3 amino acids by Cowan-Whit
Page 67 and 68: Chapter 3 Figure 15 C: Represents t
Page 69 and 70: Chapter 3 Figure 16 A: Represents t
Page 71 and 72: Chapter 3 The polarity value for th
Page 73 and 74: Chapter 3 Figure 17: Represents the
Page 77 and 78: Chapter 3 published papers with mod
Page 79 and 80: Chapter 3 ligands revealed less dif
Page 81 and 82: Chapter 3 3.2. Influence of the dif
Page 83 and 84: Chapter 3 Table 10: The chemistry o
Page 85 and 86: Chapter 3 where approximately 50% o
Page 87 and 88: Chapter 3 3.2.3.2. The electrophore
Page 89 and 90: Chapter 3 Figure 22: Represent 2-D
Page 91 and 92: Chapter 3 Table 12. List of the ide
Page 93 and 94: Chapter 3 Table 14. List of the ide
Page 95 and 96: Chapter 3 r 2 values from the data
Page 97 and 98: Chapter 3 Figure 24 C: Represents t
Page 99 and 100: Chapter 3 approximately 47%. The me
Page 101 and 102: Chapter 3 Figure 25 B: Represents t
Page 103: Chapter 3 values have been reported
Page 109 and 110: Chapter 3 among the adsorbents were
Page 111 and 112: Chapter 3 3.3. Protein distribution
Page 113 and 114: Chapter 3 3.3.2.2. Protein distribu
Page 115 and 116: Chapter 3 Figure 29 B: Represent th
Page 117 and 118: Chapter 3 When the number of smalle
Page 119 and 120: Chapter 3 and hydrophobic interacti
Page 121 and 122: Chapter 4 CONCLUSIONS
Page 123 and 124: Chapter 4 • The base supports rev
Page 125 and 126: Chapter 4 additional understanding
Page 127 and 128: References 13. Lienqueo, M.E.; Lese
Page 129 and 130: References 32. Salgado, J.C.; Rapap
Page 131 and 132: References 52. B.H.J., H. Accessibl
Page 133 and 134: References 71. Xindu, G. and Regnie
Page 135 and 136: References 91. Alonso-Orgaz, S.; Mo
Page 137 and 138: References 112. Gu, Z. and Glatz, C
Page 139: References 132. Reubsaet, J.L.E. an
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Thesis final - after defense-7 - Jacobs University

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