Thesis final - after defense-7 - Jacobs University

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.
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