Thesis final - after defense-7 - Jacobs University

Chapter 3
context of HIC based on the exposed amino acid residues of a three dimensional protein
structure. The ASH of a protein has a direct proportionality to the exposed hydrophobic
surface area on the protein surface. The three crystal structures identified in this work have
been compared for their hydrophobic surface residues and elution position during
chromatography (Figure 9). The crystal structures of the enzymes were obtained using
chimera as a visualization tool (http://www.cgl.ucsf.edu/chimera). The protein such as Serine
threonine protein kinase (A) has very less hydrophobic surface residues in comparison to the
protein Ubiquitin carboxyl terminal hydrolase 4 (B). Due to this reason, the protein A was
eluted earlier than protein B during chromatography. In the same way, the protein B has less
hydrophobic surface residues as compared to the protein Phosphogycerate mutase 1 (C). Due
to the less hydrophobic residues, the protein B was eluted earlier than protein C. There are
several other important enzymes identified at different elution ranges and can be analyzed at
the molecular level for their hydrophobic content and corresponding chromatographic
behavior. These surface hydrophobic residues can be quantified in terms of ASH from the
three dimensional structure of a protein utilizing MATLAB and STRIDE as computational
tools. ASH has been widely exploited to predict the retention times of model proteins during
HIC (103). However, all the above mentioned properties have been never studied in relation
to the host derived proteins. Therefore, the above mentioned properties were investigated for
their correlation with the chromatographic behavior and potential to differentiate among the
ligands. This work was planned to provide the data from a real bioprocess for designing the
purification model of the recombinant proteins utilizing an in silico approach.
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