Spy chemistry‐enabled protein directional immobilization and protein purification

Abstract

Site‐directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity, which is advantageous for many applications. Here, we report a facile, specific, and efficient strategy based on the SpyTag‐SpyCatcher chemistry. Two SpyTag‐fused model proteins, that is, the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl‐7‐aminocephalosporanic acid acylase, were easily immobilized onto a SpyCatcher‐modified resin directly from cell lysates, with activity recoveries in the range of 85–91%. This strategy was further adapted to protein purification, which proceeded through the selective capture of the SpyCatcher‐fused target proteins by a SpyTag‐modified resin, with the aid of an intein to generate authentic N‐termini. For two model proteins, that is, RFP and a variable domain of a heavy chain antibody, the yields were ∼3–7 mg/L culture with >90% purities. This approach could provide a versatile tool for producing high‐performance immobilized protein devices and proteins for industrial and therapeutic uses.