Magnetic Nanoplatforms for Covalent Protein Immobilization Based on Spy Chemistry

Abstract

Immobilization of proteins on magnetic nanoparticles (MNPs) is an effective approach to improve protein stability and facilitate separation of immobilized proteins for repeated use. Herein, we exploited the efficient SpyTag-SpyCatcher chemistry for conjugation of functional proteins onto MNPs and established a robust magnetic-responsive nanoparticle platform for protein immobilization. To maximize the loading capacity and achieve outstanding water dispersity, the SpyTag peptide was incorporated into the surface-charged polymers of MNPs, which provided abundant active sites for Spy chemistry while maintaining excellent colloidal stability in buffer solution. Conjugation between enhanced green fluorescence protein (EGFP)-SpyCatcher-fused proteins and SpyTag-functionalized MNPs was efficient at ambient conditions without adding enzymes or chemical cross-linkers. Benefiting from the excellent water dispersity and interface compatibility, the surface Spy reaction has fast kinetics, which is comparable to that of the solution Spy reaction. No activity loss was observed on EGFP after conjugation due to the site-selective nature of Spy chemistry. The immobilization process of EGFP on MNPs was highly specific and robust, which was not affected by the presence of other proteins and detergents, such as bovine serum albumin and Tween 20. The MNP platform was demonstrated to be protective to the conjugated EGFP and significantly improved the shelf life of immobilized proteins. In addition, experiments confirmed the retained magnetophoresis of the MNP after protein loading, demonstrating fast MNP recovery under an external magnetic field. This MNP is expected to provide a versatile and modular platform to achieve effective and specific immobilization of other functional proteins, enabling easy reuse and storage.

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

“…The recombinant plasmids (i.e., pET21b-EGFP-SpyCatcher and pET21b-RFP-SpyCatcher, Figure S2) were constructed as described previously. 35 The confirmed plasmid was then transformed into Escherichia coli (E. coli) BL21(DE3) competent cells for the expression and purification of recombinant proteins (i.e., EGFP-SpyCatcher and RFP-SpyCatcher, Figure S3) with details in the Supporting Information. After induction, the cells were harvested and sonicated for 40 × 15 s on/off pulse cycles at 30 W. The cell lysate was then centrifuged at 13000 rpm for 15 min to remove cell debris.…”

“…At predetermined time intervals, the remaining fluorescence intensity of the conjugated and free proteins was determined as described previously. 35 We also characterized the fluorescence intensity of the protein that leached out in the supernatant solutions of MNP-EGFP or MNP-RFP during storage to better understand the potential effect of leaching. To test the robustness of the conjugated proteins toward freeze−thaw processes, the conjugated and free proteins were subjected to 3 and 6 freeze−thaw cycles, and the fluorescence intensity was monitored before and after treatments.…”

“…SpyTag-functionalized MNPs that contained numerous SpyTag handles on polymer shell for SpyCatcher conjugation (Figure S1) were produced based on the reaction between allyl-modified Fe 3 O 4 @SiO 2 @PQDMAE-MA and cysteine-terminated SpyTag via thiol−ene click chemistry. 35 The conjugated MNP products, MNP-EGFP and MNP-RFP, after thorough washes showed a green and red fluorescence shell structure around the dark magnetic core, respectively (Figure 1a). MNP-EGFP and MNP-RFP also exhibited strong fluorescence intensities (Figure 1b).…”

“…On the contrary, we recently designed a SpyTag-functionalized MNP with abundant hydrophilic polymers that carry a high density of SpyTag handles. 35 The MNP maintains high water dispersibility in different aqueous media (i.e., no aggregation) and can achieve high protein loadings (∼75 μg-protein/mg-MNP). Also, protein-conjugated MNPs retained good magnetic responsiveness, enabling a facile protein recovery under an external magnetic force.…”

Fabrication of multifunctional metal–organic frameworks nanoparticles via layer-by-layer self-assembly to efficiently discover PSD95-nNOS uncouplers for stroke treatment

“…In view of the biological feasibility of this method, His tagged nNOS would selectively bind nNOS to MOFs and then specifically couple with PSD95 to establish a model for screening PSD95-nNOS uncouplers in vitro, meanwhile, the combination of PSD95-nNOS-MOF also provides an approach of purifying nNOS and PSD95 from the crude protein solution. In addition, magnetic separation can simplify the operation and speed up the separation of samples for repeated use [ 31 ]. Therefore, magnetic-MOF is first designed to bind His-tagged nNOS and then with PSD95 to construct the multifunctional nanoparticles.…”