Site-Specific and Tunable Co-immobilization of Proteins onto Magnetic Nanoparticles via Spy Chemistry.

Co-immobilization of multiple proteins onto one nanosupport has large potential in mimicking natural multiprotein complexes and constructing efficient cascade biocatalytic systems. However, control of different proteins regarding their spatial arrangement and loading ratio remains a big challenge, and protein co-immobilization often requires the use of purified proteins. Herein, built upon our recently designed SpyTag-functionalized magnetic nanoparticles (MNPs), we established a modular MNP platform for site-specific, tunable, and cost-effective protein co-immobilization. SpyCatcher-fused enhanced green fluorescent protein (i.e., EGFP-SpyCatcher) and mCherry red fluorescent protein (i.e., RFP-SpyCatcher) were designed and conjugated on MNPs, and the immobilized proteins showed 3-7-fold enhancement in storage stability and greatly improved stability against the freeze-thaw process compared to free proteins. The protein-conjugated MNPs also retained desirable colloidal stability and magnetic responsiveness, enabling facile proteins' recovery. Also, one-pot co-immobilization of the two proteins could be fine-tuned with their feed ratios. In addition, MNPs could selectively and efficiently co-immobilize both SpyCatcher-fused proteins from combined cell lysates without purification, offering a convenient and cost-effective approach for multiprotein immobilization. This MNP platform provides a facile and efficient tool to construct bionano hybrid materials (i.e., protein-based MNPs) and multiprotein systems for a variety of industrial and green chemistry applications.