Thesis final - after defense-7 - Jacobs University

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Chapter 1 contaminants which have close similarity to the target protein in the host cell proteome (19). In order to do so, one has to design the experiments to analyze the adsorption affinity of the adsorbents with a natural host. The first report about HIC was done by Sheppard and Tiselius observing the retention of dye in the presence of sulfate and phosphate solutions and was called salting out chromatography (34). Afterwards, Shalteil and Er-el used the term hydrophobic chromatography, Hofstee named the method as hydrophobic adsorption chromatography (34, 35). Finally, Hjerten in 1973 described the method as HIC (36). Porath et al. discovered that hydrophobic adsorption was enhanced by using salts like sodium chloride or phosphate chloride and named the method as salt promoted adsorption chromatography (34). A typical chromatogram obtained during my experiments has been presented in Figure 2. To facilitate the hydrophobic interactions and adsorption of proteins, it is important to load the proteins on a column with a buffer of high salt concentrations, followed by elution with a linear or a stepwise decrease in the salt concentration (37). Linear gradient can be applied during chromatographic separation of biological mixtures to separate the proteins of diverse nature. After sample application, the proteins with no binding affinity will elute in flow through and washing step. The proteins bound with the adsorbent will elute according to their binding capabilities and the extent of protein hydrophobicity. The proteins with less surface hydrophobic residues will elute at the start of the chromatography. In contrast, the proteins with high hydrophobic residues on the surface will be tightly bound and will elute at the end of the chromatography. The highly hydrophobic proteins are usually tightly bound to the column and can be removed by salt free buffer or 20% ethanol. As the surface hydrophobicity of the proteins is increasing, the retention time of the proteins will increase accordingly during chromatography. 6
Chapter 1 Salt conc. decreasing (Gradient elution) Sample application. Abs. 280 nm Unbound proteins elute before gradient. Tightly bound proteins elute in salt free conditions Column Volume (CV) Figure 2: A typical HIC chromatogram describing the chromatographic fractionation during HIC. 1.4. Factors affecting protein chromatography behavior in HIC 1.4.1. Effect of mobile phase parameters The mobile phase parameters affecting the protein retention are usually ionic strength, type of salts and buffer pH. Protein adsorption on hydrophobic adsorbents is usually favored by moderately high salt concentrations. The concentration of the salt should be according to the binding strength of the proteins and adsorbents and below the concentration causing precipitation of proteins. The optimum concentrations of salts are usually between 0.75 M and 2.0 M with ammonium sulfate and 1.0 M to 4.0 M with sodium chloride (27). The different types of salts can be divided into kosmotrophic and chaotrophic salts depending on their ability of hydrophobic interactions. The chaotrophic salts (magnesium sulfate and magnesium chloride) have less capability to bind water molecules which increases the inclusion of water molecules on the protein and ligand surface and thus decreases hydrophobic interactions 7
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 and 14: Chapter 1 cell proteome to facilita
Page 15: Chapter 1 proteins. It is this diff
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
Page 65 and 66: 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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