Small-Molecule Affinity-Based Matrices for Rapid Protein Purification
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Affinity chromatography, the method of purifying target proteins from complex mixtures using immobilized affinity ligands on chromatographic supports, is perhaps the most common of all affinity techniques (
1
–
3
). Many affinity chromatography systems are comprised of activated supports requiring direct ligand-coupling procedures that can be complex, time-consuming, and result in low- or variable-capacity columns supporting immobilized ligands with poor activity (
2
–
5
). We describe here a method that improves this technology by using a small-molecule based chemical affinity technology (
6
) to quickly and easily prepare high-capacity affinity columns supporting functionally active capture ligands for purifying proteins from crude mixtures (
7
,
8
). This innovation is based on the specific interaction between two, non-biological, small molecules, phenyl-diboronic acid (PDBA) and salicylhydroxamic acid (SHA) (
see
Fig. 1 ).
Fig. 1.
PDBA:SHA Chemical Affinity System. The reaction of phenyldiboronic acid (PDBA) with salicylhydroxamic acid (SHA). The SHA is covalently anchored to the surface of crosslinked agarose chromatography media and a PDBA derivative is conjugated to a protein of choice.
