Investigation of Protein Binding Affinity in Chromatographic Systems

The design of novel bioseparations systems is requires a better understanding of the interactions between the protein and resin surfaces. Protein charge ladders and homologous mutant libraries have been employed successfully in the study of protein binding to cation exchange surfaces. Lysozyme charge ladders were generated through the partial acetylation of surface lysines with acetic anhydride and the homologous protein mixture was separated on a cation exchanger using a linear gradient of salt. The chromatogram showed large variations in the retention time of each variant (Fig. 1). Analysis of column eluent fractions with Capillary Zone Electrophoresis (CZE) and direct infusion Electrospray ionization (ESI) revealed the presence of variants with different surface net charge coeluting off the column. Some variants with less surface positive charge were retained on the column longer than other variants that had more surface positive charge (Fig. 2). Both phenomena are highly indicative of preferred binding regions on the surface and the modification to the charges at these regions was responsible for the unique elution trends observed. Ongoing investigations involving enzyme digest mass spectrometry are being carried out to determine the exact sites of chemical surface modifications and their effect on protein retention.

Single site mutations involving both charge neutralization and reversal were performed at different locations on the surface of Cold Shock Protein B (CspB). Each mutant was analyzed on a cation exchange resin and their elution time under a linear salt gradient was observed. The general elution trend showed that mutants with more surface positive charge were bound more strongly onto the column (Fig. 3), but the difference in elution times within each class of mutant hints at the strong possibility of binding orientation changes arising as a result of the mutation. Analysis of electrostatic potential (EP) maps of the native protein and the mutants showed the different extents of alteration to the EP of the mutants which were responsible for the change in the binding orientation of the protein and ultimately the retention behavior that was observed (Fig. 4a,b,c).

Investigations into the interactions of proteins with multi-modal chromatographic reins with a variety of experimental techniques are also underway.

Fig 1. Elution Profile of a Lysozyme Charge Ladder mixture on a Protein-PAK SP8HR cation exchange column

Fig 2. Map showing identities of variants present in cation exchange eluent fractions.

Fig 3. Elution order of CspB Mutants

Fig 4a,b,c Change in electrostatic map as a result of mutations at residue 50