Synthesis of Photocleavable Biotinylated Ligands and Application for Affinity Chromatography

Summary

A method for affinity purification based on the high-affinity interaction of biotin-streptavidin and elution by irradiation with UV light was developed. As an example of this approach a biotinylated derivative of the peptide hormone cholecystokinin (CCK-8s) containing a photocleavable o-nitrobenzylester group was synthesized. The analog retained high affinity to both CCK receptors and antiCCK antibodies. It bound to a streptavidin-agarose affinity matrix and was subsequently released by irradiation with UV light of wavelengths >320 nm. For affinity purification of specific antibodies, the modified CCK-8s was incubated with anti-CCK antiserum, and the complex was subsequently passed over a streptavidin-agarose affinity matrix. After washing, the bound antibodies were eluted by photocleavage of the affinity ligand. The eluted antibodies showed essentially unchanged binding characteristics. The approach may prove to be generally useful in the isolation of labile proteins in an intact form.