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

Abstract

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 coimmobilization. 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 proteinconjugated 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.

Lanmodulin-Functionalized Magnetic Nanoparticles as a Highly Selective Biosorbent for Recovery of Rare Earth Elements

“…Protein expression was conducted based on a previously published method, 22 except that 100 μg/mL ampicillin was added into the LB medium. The collected cells were disrupted by sonication following our established method, 32 and LanM-SpyCatcher was purified using a HisPur Ni-NTA Purification Kit (Thermofisher, USA) following the manufacturer's instructions. The eluted protein was concentrated using the Vivaspin 6 centrifugal concentrator (GE Healthcare, USA, 10 kDa MWCO) and confirmed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining.…”

Sequential Co-Immobilization of Enzymes on Magnetic Nanoparticles for Efficient l-Xylulose Production

“…Significant disadvantages include (i) lower activity and reaction rates compared to free enzymes, (ii) surplus cost of immobilization supports, (iii) fouling, and (iv) disposal of exhausted immobilized biocatalysts through incineration or environmental issues due to material toxicity [ 1 , 4 , 8 , 9 , 10 ]. Such issues can potentially be solved by the selective use of immobilization methods, supports, and protein engineering-based approaches in specific enzyme immobilization [ 11 ]. In addition, multi-enzyme immobilization has proven to be more beneficial than free-enzyme mixtures for efficient bioconversion or bioremediation application [ 8 , 12 , 13 , 14 ].…”

Spontaneous and site-specific immobilization of PNGase F via spy chemistry

“…29,30 With these intriguing features, Spy chemistry, since it was reported in 2012, 28 has been used in many applications, including protein ligation and protein immobilization in solid materials, including hydrogels, 31 agarose, 27 silica, 32 gold nanoparticles, 33 and magnetic nanoparticles. 34,35 Therefore, Spy chemistry is an excellent approach to immobilize PNGase F, minimizing the risk of PNGase F inactivation in traditional covalent immobilization processes.…”