An affinity-based strategy for the design of selective displacers for the chromatographic separation of proteins.
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.
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Srinavya
S
Vutukuru
Sandesh D
SD
Kate
Scott A
SA
McCallum
Christopher J
CJ
Morrison
Steven M
SM
Cramer
Ravi S
RS
Kane
D000818:Animals; D007611:Aprotinin / Q000302:isolation & purification; D001360:Avidin / Q000302:isolation & purification; D001710:Biotin / Q000737:chemistry; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D006736:Horses; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009113:Muramidase / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*
D011506:Proteins; D001360:Avidin; D001710:Biotin; D007611:Aprotinin; D009113:Muramidase