Premium
Investigation of protein binding affinity and preferred orientations in ion exchange systems using a homologous protein library
Author(s) -
Chung Wai Keen,
Hou Ying,
Freed Alexander,
Holstein Melissa,
Makhatadze George I.,
Cramer Steven M.
Publication year - 2008
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22100
Subject(s) - mutant , chemistry , ion chromatography , charge density , amino acid , affinity chromatography , biophysics , chromatography , biochemistry , biology , physics , quantum mechanics , gene , enzyme
A library of cold shock protein B (CspB) mutant variants was employed to study protein binding affinity and preferred orientations in cation exchange 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. While the expected trends were observed with increasing or decreasing positive charge on the protein surface, the degree of change was a strong function of the location and microenvironment of the mutated amino acid. Quantitative structure–property relationship (QSPR) models were generated using a support vector regression technique that was able to give good predictions of the retention times of the various mutants. Molecular descriptors selected during model generation were used to elucidate the factors affecting protein retention. Electrostatic potential maps were also employed to provide insight into the effects of protein surface topography, charge density and charge distribution on protein binding affinity and possible preferred binding orientations. The use of this protein mutant library in concert with the qualitative and quantitative analyses presented in the article provides an improved understanding of protein behavior in ion exchange systems. Biotechnol. Bioeng. 2009; 102: 869–881. © 2008 Wiley Periodicals, Inc.