An Affinity-Based Strategy for the Design of Selective Displacers for the Chromatographic Separation of Proteins

Abstract

We describe an affinity-based strategy for designing selective protein displacers for the chromatographic purification of proteins. To design a displacer that is selective for a target protein, we attached a component with affinity for the target protein to a resin-binding component; we then tested the ability of such displacers to selectively retain the target protein on a resin relative to another protein having a similar retention time. In particular, we synthesized displacers based on biotin, which selectively retained avidin as compared to aprotinin on SP Sepharose high performance resin. In addition, we have extended this approach to develop an affinity-peptide-based displacer that discriminates between lysozyme and cytochrome c. Here, a selective displacer was designed from a lysozyme-binding peptide that had been identified and optimized previously using phage-display technology. Our results suggest a general strategy for designing highly selective affinity-based displacers by identifying molecules (e.g., peptides) that bind to a protein of interest and using an appropriate linker to attach these molecules to a moiety that binds to the stationary phase.

Surface Plasmon Resonance Spectroscopy-Based High-Throughput Screening of Ligands for Use in Affinity and Displacement Chromatography

“…We previously described a rational approach for designing selective affinitybased displacers by combining a protein-binding moiety with a stationary phase-binding component. 39 Because ligands 3, 4, and 11 have a greater affinity for lysozyme than for cytochrome c, and also have a positively charged amine group which could serve as a stationary phase-binding component, we reasoned that they may serve as selective displacers (displacing cytochrome c to a greater extent than lysozyme). We used ligands 1 and 7 as controls.…”

“…Encouraged by the results presented above, we next decided to test whether the ligands identified by screening might also be useful as chemically selective displacers for displacement chromatographya technique that is operationally simple and offers high yield and product concentration. − Chemically selective displacers function by retaining a protein of interest on a stationary phase while displacing other proteins. We previously described a rational approach for designing selective affinity-based displacers by combining a protein-binding moiety with a stationary phase-binding component . Because ligands 3 , 4 , and 11 have a greater affinity for lysozyme than for cytochrome c , and also have a positively charged amine group which could serve as a stationary phase-binding component, we reasoned that they may serve as selective displacers (displacing cytochrome c to a greater extent than lysozyme).…”