Self‐assembling protein nanocages for modular enzyme assembly by orthogonal bioconjugation

Abstract

The wide variety of enzymatic pathways that can benefit from enzyme scaffolding is astronomical. While enzyme co‐localization based on protein, DNA, and RNA scaffolds has been reported, we still lack scaffolds that offer well‐defined and uniform three‐dimensional structures for enzyme organization. Here we reported a new approach for protein co‐localization using naturally occurring protein nanocages as a scaffold. Two different nanocages, the 25 nm E2 and the 34 nm heptatitis B virus, were used to demonstrate the successfully co‐localization of the endoglucanase CelA and cellulose binding domain using the robust SpyTag/SpyCatcher bioconjugation chemistry. Because of the simplicity of the assembly, this strategy is useful not only for in vivo enzyme cascading but also the potential for in vivo applications as well.

Incorporation of Endosomolytic Peptides with Varying Disruption Mechanisms into EGFR-Targeted Protein Conjugates: The Effect on Intracellular Protein Delivery and EGFR Specificity in Breast Cancer Cells

“…Endosomolytic peptides have shown some ability to trigger cell internalization, which can hinder targeting specificity. For instance, Aurein 1.2 enhanced the uptake of fused protein nanocarriers in prior work . Here, fusing a single Aurein 1.2 peptide to 4GE11-mCherry-SC did not significantly increase uptake, indicating that protein internalization remained primarily EGFR-mediated.…”

“…For instance, Aurein 1.2 enhanced the uptake of fused protein nanocarriers in prior work. 74 Here, fusing a single Aurein 1.2 peptide to 4GE11-mCherry-SC did not significantly increase uptake, indicating that protein internalization remained primarily EGFR-mediated. Despite maintaining some EGFR specificity, GALA decreased the internalization of 4GE11- mCherry-SC in IBC SUM149 cells.…”

Efficient Synthesis of Phycocyanobilin by Combinatorial Metabolic Engineering in Escherichia coli

“…Since the application of different promoters and copy number of plasmids cannot further improve PCB synthesis, another strategy was utilized based on the spatial organization of HO and PcyA and the regulation of their ratio because these two enzymes natively form a 1:2 complex with Fd to synthesize PCB in cyanobacteria, respectively . Although several technologies for organizing metabolic multienzymes, including the DNA scaffold, protein scaffold, and self-assembling nanocages, have been used to enhance the catalytic efficiency of intracellular cascade reactions or control metabolic flux, the DNA scaffold was selected to minimize the adverse impact of extra-heterologous proteins on the expression of HO and PcyA. Hence, three engineered strains with various regulated ratios of HO1 and PcyA (1:1, 2:1, and 1:2) were constructed (P13 strain: harboring pRSFDuet-T7 lac - ADB 1- ho 1 T -T7 lac - ADB 2- pcyA S and pACYCDuet-T7 lac -scaffold1-scaffold2 plasmids; P14 strain: harboring pRSFDuet-T7 lac - ADB 1- ho 1 T -T7 lac - ADB 2- pcyA S and pACYCDuet-T7 lac -scaffold1-scaffold2-scaffold1 plasmids; P15 strain: harboring pRSFDuet-T7 lac - ADB 1- ho 1 T -T7 lac - ADB 2- pcyA S and pACYCDuet-T7 lac -scaffold2-scaffold1-scaffold2 plasmids).…”

EGFR Ligand Clustering on E2 Bionanoparticles for Targeted Delivery of Chemotherapeutics to Breast Cancer Cells

“…26 This approach allowed for modular, site-specific surface decoration of E2 for antibody sensing, 27 purification, 26 and enzyme scaffolding. 28 We hypothesized that the SC/ST approach also would be an efficient strategy for decorating E2 with targeting ligands to enhance cell specific internalization.…”

“…To utilize SC/ST chemistry for surface decoration, the 13-amino acid ST peptide was fused to the N-terminus of each of the 60 subunits . This approach allowed for modular, site-specific surface decoration of E2 for antibody sensing, purification, and enzyme scaffolding . We hypothesized that the SC/ST approach also would be an efficient strategy for decorating E2 with targeting ligands to enhance cell specific internalization.…”