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The tetrameric protein streptavidin has found ubiquitous use in a wide variety of applications due to its ability to bind biotin molecules with remarkable affinity (K d of 10 − 14 M). However, for applications that require equimolar conjugation, streptavidin and its four active biotin-binding sites can be difficult to employ. To address this need, a monovalent variant of streptavidin comprising one wild-type (biotin binding) and three mutant (non-binding) streptavidin monomers has emerged that conserves the characteristic binding affinity of the wild-type protein but with only a single biotin recognition pocket. While monovalent streptavidin has found broad utility in biotechnology, the difficult nature of its production—involving its retrieval from insoluble inclusion bodies upon recombinant expression—has limited its availability as a powerful biological tool. Here, we present an improved method for monovalent streptavidin production that circumvents critical challenges by implementing a protein solubility partner to streamline isolation, minimize laborious hands-on procedures, and achieve exceptional protein purity and yield. First, we describe plasmid design and recombinant expression of maltose binding protein fusions with both wild-type and mutant streptavidin monomers. Next, we outline the chromatographic steps to isolate these products independently from cell lysate and cleave the monomers enzymatically from their fusion partner. Finally, we detail the folding, purification, and functional validation of the resulting tetrameric streptavidin consisting of one wild-type and three mutant monomers. This scalable workflow achieves exceptional yield (7 mg per L of culture) and purity (approximately 95%) of monovalent streptavidin.

The fast development of single-particle cryogenic electron microscopy (cryo-EM) has made it more feasible to obtain the 3D structure of well-behaved macromolecules with a molecular weight higher than 300 kDa at ~3 Å resolution. However, it remains a challenge to obtain the high-resolution structures of molecules smaller than 200 kDa using single-particle cryo-EM. In this work, we apply the Cs-corrector-VPP-coupled cryo-EM to study the 52 kDa streptavidin (SA) protein supported on a thin layer of graphene and embedded in vitreous ice. We are able to solve both the apo-SA and biotin-bound SA structures at near-atomic resolution using single-particle cryo-EM. We demonstrate that the method has the potential to determine the structures of molecules as small as 39 kDa. It remains a challenge to obtain high-resolution structures of molecules smaller than 200 kDa using single particle cryo-EM. Here, the authors apply the Cs-corrector-VPP coupled cryo-EM and solve structures of the 52 kDa streptavidin (SA) protein at near-atomic resolution.

Enzymes provide an exquisitely tailored chiral environment to foster high catalytic activities and selectivities, but their native structures are optimized for very specific biochemical transformations. Designing a protein to accommodate a non-native transition metal complex can broaden the scope of enzymatic transformations while raising the activity and selectivity of small-molecule catalysis. Here, we report the creation of a bifunctional artificial metalloenzyme in which a glutamic acid or aspartic acid residue engineered into streptavidin acts in concert with a docked biotinylated rhodium(III) complex to enable catalytic asymmetric carbon-hydrogen (C-H) activation. The coupling of benzamides and alkenes to access dihydroisoquinolones proceeds with up to nearly a 100-fold rate acceleration compared with the activity of the isolated rhodium complex and enantiomeric ratios as high as 93:7.

Existing strategies use bifunctional chimaeras to mediate extracellular protein degradation. However, these strategies rely on specific lysosome-trafficking receptors to facilitate lysosomal delivery, which may raise resistance concerns due to intrinsic cell-to-cell variation in receptor expression and mutations or downregulation of the receptors. Another challenge is establishing a universal platform applicable in multiple scenarios. Here, we develop MONOTAB (MOdified NanOparticle with TArgeting Binders), a plug-and-play monofunctional degradation platform that can drag extracellular targets into lysosomes for degradation. MONOTAB harnesses the inherent lysosome-targeting ability of certain nanoparticles to obviate specific receptor dependency and the hook effect. To achieve high modularity and programmable target specificity, we utilize the streptavidin-biotin interaction to immobilize antibodies or other targeting molecules on nanoparticles, through an antibody mounting approach or by direct binding. Our study reveals that MONOTAB can induce efficient degradation of diverse therapeutic targets, including membrane proteins, secreted proteins, and even extracellular vesicles. Existing methods use bifunctional chimaeras for extracellular protein degradation but rely on specific lysosome-targeting receptors, raising resistance issues. Here, the authors develop MONOTAB, a monofunctional platform that degrades extracellular proteins and vesicles without receptor dependency.

Proximity biotinylation, which utilizes various biotin ligating enzymes (BioID, TurboID, etc.), is widely used as a powerful tool for identifying novel protein–protein interactions. However, this method has a significant limitation: the use of streptavidin on beads for enriching biotinylated proteins often results in a high background of peptides from streptavidin itself, which interferes with identification by peptide mass fingerprinting. This limitation makes it practically impossible to study samples containing a small amount of material, such as individual insect tissues. In this study, we compared different precipitation and elution conditions for the purification of biotinylated proteins from protein extracts of Drosophila melanogaster S2 cells. We found that biotinylated proteins can be purified using anti-biotin antibodies, although with lower efficiency than streptavidin-based resin. We also demonstrated that protease-resistant streptavidin (prS), previously tested in mammalian cells, can be used effectively to purify biotinylated proteins from tissues of D. melanogaster. In our experiments, prS showed precipitation efficiency comparable to regular streptavidin but generated a lower background in peptide fingerprinting. To further demonstrate the applicability of prS for studying protein–protein interactions in D. melanogaster tissues, we carried out experiments to identify interaction partners of the ecdysone receptor (EcR) in D. melanogaster ovarian tissue using TurboID-based proximity biotinylation. As a result, EcR was found to interact with both previously described and novel protein partners in Drosophila ovaries.

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3. Cyclization provides a general strategy for improving peptide proteolytic stability, cell membrane permeability and target binding affinity. Here the authors develop a cyclopropenone-based proximity-driven chemical linker for the site-specific cyclization of phage-displayed peptides.

Cyclic peptides constitute an important drug modality since they offer significant advantages over small molecules and macromolecules. However, access to diverse chemical sets of cyclic peptides is difficult on a large library scale. DNA-encoded Chemical Libraries (DELs) provide a suitable tool to obtain large chemical diversity, but cyclic DELs made by standard DEL implementation cannot efficiently explore their conformational diversity. On the other hand, dual-display Encoded Self-Assembling Chemical (ESAC) Libraries can be used for modulating macrocycle flexibility since the two displayed peptides can be connected in an incremental fashion. In this work, we construct a 56 million dual-display ESAC library using a two-step cyclization strategy. We show that varying the level of conformational restraint is essential for the discovery of specific ligands for the three protein targets thrombin, human alkaline phosphatase and streptavidin. Cyclic peptides are an important drug modality but access to diverse chemical sets of cyclic peptides is difficult on a large library scale. Here, the authors use DNA-encoded library (DEL) technology for the dual-display of two peptidic sub-libraries at the extremities of DNA heteroduplexes and show that varying the level of conformational restraint is essential for the discovery of specific ligands for the three protein targets.

Circulating tumor cells (CTCs) are important biomarkers for cancer diagnosis and treatment monitoring. However, their scarcity limits their utility as current enrichment techniques are hampered by low volume throughput and/or the inability to capture CTCs with low target antigen densities. Our group previously reported a device capable of processing samples in a flow manner using an optimized Halbach array to enhance the capture of low EpCAM-expressing cells (Flow-through Immunomagnetic CTC Enrichment system). In this study, we tested the capture efficiency of eight commercially available streptavidin magnetic beads using this device to identify the most suitable bead. Results indicate that using this system, the best-performing magnetic beads are in the ~ 100 to ~ 150 nm size range. Considering the combination of binding efficiency and final sample purity, we found that among the beads tested in combination with biotinylated anti-EpCAM, MojoSort Streptavidin Nanobeads performed the best, with high capture efficiencies for both the high EpCAM expressing LNCaP and low EpCAM expressing PC3–9 cell lines. For CTC enrichment from the blood of cancer patients, reducing the number of WBCs co-enriched with these beads will be essential, especially when processing large-volume samples acquired, for instance, through diagnostic leukapheresis to overcome the limitations caused by the scarcity of CTCs.

Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an ‘all in one’ solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

The concept of point of darkness has received much attention for biosensing based on phase-sensitive detection and perfect absorption of light. The maximum phase change is possible at the point of darkness where the reflection is almost zero. To date, this has been experimentally realized using different material systems through the concept of topological darkness. However, complex nanopatterning techniques are required to realize topological darkness. Here, we report an approach to realize perfect absorption and extreme phase singularity using a simple metal-dielectric multilayer thin-film stack. The multilayer stack works on the principle of an asymmetric Fabry–Perot cavity and shows an abrupt phase change at the reflectionless point due to the presence of a highly absorbing ultrathin film of germanium in the stack. In the proof-of-concept phase-sensitive biosensing experiments, we functionalize the film surface with an ultrathin layer of biotin-thiol to capture streptavidin at a low concentration of 1 pM. Optical sensors generally rely on abrupt phase changes to detect the presence of an analyte, but oftentimes they require complex nanostructures. Here, Sreekanth et al. use a simple asymmetric thin-film multilayer stack to demonstrate a point of darkness and phase singularity to develop a sensitive biosensor.