A polyvalent antibody-binding lumazine synthase protein cage nanoparticle (ABD–AaLS) is constructed by genetically fusing lumazine synthase and antibody-binding domains. ABD–AaLS effectively displays targeting antibodies in an orientation-controlled manner.

With high brightness and photostability, quantum dots (QDs) are potent probes for long‐term imaging of dynamic cell surface proteins, but practical methods to covalently label QDs to target proteins for stable imaging are largely lacking. Here, a small covalent‐bond forming protein (Covalent‐avidin)/peptide pair is introduced, which provides a recombinant protein‐based rapid and covalent QD labeling strategy. Covalent‐avidin is constructed by optimized fusion of circular permuted monomeric avidin to SpyCatcher, which forms an isopeptide bond with the SpyTag peptide. Covalent‐avidin‐conjugated QDs allow for strong and irreversible QD labeling to the biotinylated SpyTag‐fused adrenergic receptor on live cells in 2 min. In addition, QDs with only a minimum number of conjugated Covalent‐avidin show more stable receptor labeling than commercially available streptavidin‐conjugated QDs, also with minimal unwanted clustering of labeled receptors. Monomeric Covalent‐avidin will be a valuable protein linker for diverse other nanolabeling structures with beneficial properties such as covalent linkages and facile valency control.

Supramolecular protein assemblies have garnered considerable interest due to their potential in diverse fields with unrivaled attainable functionalities and structural accuracy. Despite significant advances in protein assembly strategies, inserting long linkers with varied lengths and rigidity between assembling protein building blocks remains extremely difficult. Here we report a series of green fluorescent protein (GFP) oligomers, where protein building blocks were linked via two independent peptide strands.Assembling protein units for this two-peptide assembly were designed by flopped fusion of three selfassembling GFP fragments with two peptide linkers. Diverse flexible and rigid peptide linkers were successfully inserted into high-valent GFP oligomers. In addition, oligomers with one flexible linker and one rigid linker could also be fabricated, allowing more versatile linker rigidity control. Linker length could be varied from 10 amino acids (aa) even up to 76 aa, which is the longest among reported protein assembling peptide linkers. Discrete GFP oligomers containing diverse linkers with valencies between monomers to decamers were monodispersely purified by gel elution. Furthermore, various functional proteins could be multivalently fused to the present GFP oligomers. Binding assays, size exclusion chromatography, dynamic light scattering, circular dichroism, differential scanning calorimetry, and transmission electron microscopy suggested circular geometries of the GFP oligomers and showed distinct characteristics of GFP oligomers with length/rigidity varied linkers. Lastly, a surface binding study indicated that more spaced oligomeric binding modules offered more effective multivalent interactions than less spaced modules.Scheme 1 Schematic representation of expected oligomerization processes of two peptide-linked GFP oligomers. Blue, red, and gray denote GFP 10, GFP 11, and GFP 1-9, respectively. The N-and Ctermini of the monomeric building block are depicted as 'N' and 'C', respectively.This journal is