Common protein purification processes rely on noncovalent affinity binding of the target protein to resin via fusion tags, which has limitations such as leakage of target during the removal of impurities, restricting stringency of wash conditions, and often requires postpurification removal of tags. Here, we developed the Catch, Hold, and Release (C-H-R) method by bioengineering a single-use resin composed of polyhydroxybutyrate beads codisplaying two SpyCatcher (SpyC) domains and Sortase A (SrtA). This core-shell resin was assembled by engineered Escherichia coli. SpyTagged (SpyT) targets with a SrtA cleavage site after SpyT were specifically ligated to SpyC, while inducible SrtA catalyzed the release of the pure tagless target. SpyT remained covalently bound to the resin. Three diverse proteins were isolated from complex mixtures with high purity and yields. The C-H-R bioseparation method uses an innovative resin engaging a covalent link to the target and an inducible proximal SrtA as a protein purification concept.