Fig. 4 | Nature Communications

Fig. 4

From: Modular assembly of proteins on nanoparticles

Fig. 4

Hierarchical assembly of GST-SpyCatcher and SpyTag on GNPs. a Design of the conjugation strategy. Blue identifies the first layer of proteins, orange highlights the second layer. Figure is not to scale. b Equilibrium binding data of GST-SpyCatcher compared to SpyCatcher without GST. The fit to the binding model shows that GST fusion improves binding of SpyCatcher to GNPs. Data points and error bars represent average and standard deviation of three measurements respectively. c Comparison of UV–vis spectra of GNPs coated with GST-SpyCatcher or SpyCatcher before and after adding 2-ME. Whereas SpyCatcher/GNPs are destabilized by 2-ME, GST-SpyCatcher/GNPs are stable, suggesting formation of Au–S bonds. Vertical dashed lines indicate the two wavelengths previously used for calculations of the Au–S index and highlight a sharp decrease of the SPR peak at 525 nm in absence of GST. d Δd of GST-SpyCatcher/GNPs before (dark gray) and after (light gray) addition of either NEM-GST-SpyTag (left) or untagged NEM-GST (right). Tagging NEM-GST with SpyTag results in a significant increase of Δd, suggesting that a second layer of protein extensively binds to GNPs. Δd are averaged over three measurements with error bars representing standard deviation. t-test results with a 95% confidence interval are indicated by asterisk (significant, p = 0.0001) and NS (non-significant, p = 0.0991). e SDS-PAGE of proteins bound to GNPs. Lane 1: GST-SpyCatcher/GNPs before any further incubation; lane 2: GST-SpyCatcher/GNPs incubated with untagged NEM-GST; lane 3: GST-SpyCatcher/GNPs incubated with NEM-GST-SpyTag; lane 4: protein marker (relevant molecular weights are reported on the right). A band corresponding to GST-SpyCatcher-NEM-GST-SpyTag appears in lane 3, suggesting formation of the isopeptide bond between the two layers of protein corona

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