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Investigation of protein binding affinity in multimodal chromatographic systems using a homologous protein library
Authors:Wai Keen Chung  Ying Hou  Melissa Holstein  Alexander Freed  George I. Makhatadze  Steven M. Cramer
Affiliation:1. Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA;2. Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA;3. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA
Abstract:A library of cold shock protein B mutant variants was employed to examine differences in protein binding behavior in ion exchange and multimodal chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography in both systems. The majority of the proteins were more strongly retained on the multimodal cation exchange resin as compared to the traditional cation exchanger. Further, the elution order of the mutants on the multimodal resin was different from that obtained with the ion exchanger. Quantitative structure–property relationship models generated using a support vector regression technique were shown to provide good predictions for the retention times of protein mutants on the multimodal resin. A coarse-grained ligand docking package was employed to examine the various interactions between the proteins and ligands in free solution. The multimodal ligand was shown to utilize multiple interaction types to achieve stronger retention on the protein surface. The use of this protein library in concert with the qualitative and quantitative analyses presented in this paper provides an improved understanding of protein behavior in multimodal chromatographic systems.
Keywords:Multimodal chromatography   Mixed mode chromatography   Homologous protein library   Structure&ndash  property relationships   Protein&ndash  ligand interactions   Coarse-grained simulations
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