Single amino acid contributions to protein retention in cation-exchange chromatography: resolution of genetically engineered subtilisin variants

Genetically engineered proteins were used to determine the amino acid contributions of surface residues to subtilisin retention in cation-exchange chromatography. Crystallographic data were used to correlate the observed chromatographic behavior with enzymatic structure. Retention times of variants in gradient elution varied by as much as 33% compared to the wild type. The role of both charged and uncharged residues was investigated in isocratic separations and found to significantly influence protein retention in this electrostatically dominant separation method. This study demonstrates the ability of ion-exchange chromatography to discriminate between protein variants differing by a single residue in 275 amino acids.