Substrate‐Baited Nanoparticles: A Catch and Release Strategy for Enzyme Recognition and Harvesting

Abstract

The isolation of a single type of protein from a complex mixture is vital for the characterization of the function, structure, and interactions of the protein of interest and is typically the most laborious aspect of the protein purification process. In this effort, a model system was utilized to show the efficacy of synthesizing a “baited” nanoparticle to capture and recycle enzymes (proteins that catalyze chemical reactions) from crude cell lysate. Enzyme trapping and recycling was illustrated with the CARDO system, an enzyme important in bioremediation and natural product synthesis. The enzymes were baited with azide modified carbazolyl moieties attached to poly (propargyl acrylate) nanoparticles through a click transformation. MALDI-TOF and SDS-PAGE analysis indicated the single step procedure to immobilize the enzymes on the particles was capable of significantly concentrating the protein from raw lysate and sequestering all required components of the protein to maintain bioactivity. These results establish a universal model applicable to concentrating and extracting known substrate protein pairs, but it can be an invaluable tool in recognizing unknown protein-ligand affinities.

Mechanical and Anticorrosive Properties of Graphene/Epoxy Resin Composites Coating Prepared by in-Situ Method

“…Hence, there was more plastic deformation remaining. At the same time, it was found that the plasticity index of GNS-based epoxy resin coating was lower than that reported in previous works [ 16 , 21 ]. The result was attributed to good adhesion and dispersion of graphene modified by PVP in epoxy resin matrix.…”

<p>Time-dependent degradation of carbon nanotubes correlates with decreased reactive oxygen species generation in macrophages</p>

“…The black spots representing SG-CNTs inside the RAW 264.7 cells decreased after incubation for an additional 1–7 days, suggesting that some of the SG-CNTs were degraded by the RAW 264.7 cells. To quantify the amount of SG-CNT degradation by macrophages, the SG-CNTs in the cell lysate were measured using the optical absorption method 18. As shown in Figure 1B, the amount of SG-CNTs decreased by about 30% from Day 0 to Day 1; almost no further degradation occurred between Day 4 and Day 7.…”

“…To understand the relative long-term toxicity of CNTs after uptake by macrophages, we investigated CNT biodegradation and cytotoxicity for up to 7 days after uptake by two types of macrophage: the mouse macrophage cell line RAW 264.7 and primary rat Kupffer cells. Changes in the amount of intracellular CNTs over the 7 days were measured using our previously published optical absorption method18 and cytotoxicity was evaluated by measuring ROS, glutathione, cell viability, and caspase 3/7 activity. Over the 7-day test period, CNTs were partially degraded by macrophages.…”

<p>A brief review of cytotoxicity of nanoparticles on mesenchymal stem cells in regenerative medicine</p>

“…Liu et al demonstrated that silica-coated core-shell superparamagnetic iron oxide NPs co-condensed with fluorescein isothiocyanate-incorporated mesoporous silica were able to facilitate uptake by hMSCs without affecting their viability and differentiation potential at the same time 87. More meaningfully, cyanine dye-doped silica NPs were reported to label hMSCs without affecting viability, proliferation, stemness surface marker expression, and the ability to differentiate into osteocytes 88. Also, after the internalization of mesoporous silica NPs conjugated with fluorescein isothiocyanate by hMSCs, Huang et al observed that there were no obvious differences in cell viability, proliferation, and surface markers, compared to unlabeled cells 89.…”