Spy chemistry-enabled protein directional immobilization and protein purification.

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, i.e. the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl-7-aminocephalosporanic acid acylase (GA), 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, i.e. RFP and a variable domain of a heavy chain antibody (VHH), 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. This article is protected by copyright. All rights reserved.