Compact and modular bioprobe: Integrating SpyCatcher/SpyTag recombinant proteins with zwitterionic polymer-coated quantum dots.

The development of quantum dot (QD)-based modular bioprobe that has a compact size and enable a facile conjugation of various biofunctional groups is in high demand. To address this, we surface engineered QDs with zwitterion polymer ligands to have an inherent compact size and derivatized them sequentially with the recombinant proteins SpyCatcher/SpyTag (SC/ST) to use their protein ligation system. SC/ST spontaneously form one complex through the isopeptide bond between them. SC-conjugated QDs (QD-SC) were used as base building blocks. Then, ST-biomolecules were added for modular biofunctionalization. We synthesized compact sized (∼15 nm) QD-SC-ST-affibody (antibody-mimicking small protein for tumor detection) conjugates, which showed successful cell-receptor targeting. The target cell-receptor could be easily tuned by changing the type of ST-affibody. We also demonstrated that anti-human-chorionic-gonadotropin mouse IgG1 antibodies can be labeled on the QD surface by mixing QD-SC with the ST-MG1Nb (mouse-IgG1-specific protein). The immunoassay performance of the antibody-labeled QDs was evaluated using a pregnancy test kit, displaying equivalent detection sensitivity to a commercially available kit. This study proposed an innovative strategy for QD biofunctionalization in a modular manner, which can be expanded to a diverse range of colloidal particle derivatization.

Development of recombinant secondary antibody mimics (rSAMs) for immunoassays through genetic fusion of monomeric alkaline phosphatase with antibody binders.

In conventional immunoassays, a secondary antibody is used to amplify the signal generated by the binding of the primary antibody to the target analyte. Due to concerns regarding animal use and cost-inefficiency of secondary antibody productions, there is a significant demand for the development of recombinant secondary antibody mimics (rSAMs). Here, we developed rSAMs using a signal-generating enzyme, monomeric alkaline phosphatase (mALP), and antibody-binders, including monomeric streptavidin (mSA2) and mouse IgG1- or rabbit IgG-binding nanobodies (MG1Nb or RNb). The mALP-MG1Nb, mALP-RNb, and mALP-mSA2 were genetically constructed and produced in large quantities using bacterial overexpression systems, which reduced manufacturing costs and time without the use of animals. Each rSAM exhibited high and selective binding to its respective primary antibody, generating linear band signals corresponding to the amounts of target analytes in western blots. The rSAMs also successfully generated sigmoidal signal curves that increased as the sample concentration increased. Moreover, they generated stronger signals than conventional ALP-conjugated secondary antibodies and SA, particularly in the medium to high sample concentration range, in both indirect and sandwich-type indirect ELISAs at the same sample concentration. The rSAMs we developed here may provide new insights to develop novel immunoassay-based analytical and diagnostic tools.

Hybrid CRISPR/Cas protein for one-pot detection of DNA and RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as next-generation molecular diagnostics. In CRISPR-based diagnostics, Cas12 and Cas13 proteins have been widely employed to detect DNA and RNA, respectively. Herein, we developed a novel hybrid Cas protein capable of detecting universal nucleic acids (DNA and RNA). The CRISPR/hybrid Cas system simultaneously recognizes both DNA and RNA, enabling the dual detection of pathogenic viruses in a single tube. Using wild-type (WT) and N501Y mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as detection models, we successfully detected both virus strains with a detection limit of 10 viral copies per reaction without cross-reactivity. Furthermore, it is demonstrated the detection of WT SARS-CoV-2 and N501Y mutant variants in clinical samples by using the CRISPR/hybrid Cas system. The hybrid Cas protein is expected to be utilized in a molecular diagnostic method for infectious diseases, tissue and liquid biopsies, and other nucleic acid biomarkers.

Tunable Physicomechanical and Drug Release Properties of In Situ Forming Thermoresponsive Elastin-like Polypeptide Hydrogels.

With the continued advancement in the design and engineering of hydrogels for biomedical applications, there is a growing interest in imparting stimuli-responsiveness to the hydrogels in order to control their physicomechanical properties in a more programmable manner. In this study, an in situ forming hydrogel is developed by cross-linking alginate with an elastin-like polypeptide (ELP). Lysine-rich ELP synthesized by recombinant DNA technology is reacted with alginate presenting an aldehyde via Schiff base formation, resulting in facile hydrogel formation under physiological conditions. The physicomechanical properties of alginate-ELP hydrogels can be controlled in a wide range by the concentrations of alginate and ELP. Owing to the thermoresponsive properties of the ELP, the alginate-ELP hydrogels undergo swelling/deswelling near the physiological temperature. Taking advantage of these highly attractive properties of alginate-ELP, drug release kinetics were measured to evaluate their potential as a thermoresponsive drug delivery system. Furthermore, an ex vivo model was used to demonstrate the minimally invasive tissue injectability.

Highly sensitive pregnancy test kit via oriented antibody conjugation on brush-type ligand-coated quantum beads.

Lateral flow assays (LFA) enable development of portable and rapid diagnostic kits; however, their capacity to detect low levels of disease markers remains poor. Here, we report a highly sensitive pregnancy test kit as a proof of concept, by combining brush-type ligand-coated quantum beads (B-type QBs) and nanobody, which can control the antibody orientation and enhance sensitivity. The brush-type ligand provided excellent dispersion stability and high-binding capacity toward antibody. Fc-binding nanobody increased the antigen-binding capacity of conjugated antibodies on the B-type QBs. To facilitate convenient acquisition of the LFA results, we developed a smartphone-based reader with a 3D-printed optical imaging module, and validated the diagnostic performance of the sensing platform. The pregnancy test kit achieved a 5.1 pg mL-1 limit of detection, corresponding to the levels for early-stage detection of heart disease and malaria. Our LFA application can potentially be expanded to diagnosis other diseases by simply changing the antibody pair in the kit.

Development of antibody against drug-resistant respiratory syncytial virus: Rapid detection of mutant virus using split superfolder green fluorescent protein-antibody system.

Respiratory syncytial virus (RSV) infections are associated with severe bronchiolitis or pneumonia. Although palivizumab is used to prevent RSV infections, the occurrence of palivizumab-resistant RSV strains is increasing, and these strains pose a threat to public health. Herein, we report an antibody with affinity to the S275F RSV antigen, enabling the specific detection of palivizumab-resistant RSV strains. Experimental and simulation results confirmed the affinity of the antibody to the S275F RSV antigen. Furthermore, we developed a rapid S275F RSV antigen detection method using a split superfolder green fluorescent protein (ssGFP) that can interact with the antibody. In the presence of the mutant virus antigen, ssGFP emitted fluorescence within 1 min, allowing the rapid identification of S275F RSV. We anticipate that the developed antibody would be useful for the precise diagnosis of antiviral drug-resistant RSV strains and help treat patients with RSV infections.

Load and Display: Engineering Encapsulin as a Modular Nanoplatform for Protein-Cargo Encapsulation and Protein-Ligand Decoration Using Split Intein and SpyTag/SpyCatcher.

Protein cage nanoparticles have a unique spherical hollow structure that provides a modifiable interior space and an exterior surface. For full application, it is desirable to utilize both the interior space and the exterior surface simultaneously with two different functionalities in a well-combined way. Here, we genetically engineered encapsulin protein cage nanoparticles (Encap) as modular nanoplatforms by introducing a split-C-intein (IntC) fragment and SpyTag into the interior and exterior surfaces, respectively. A complementary split-N-intein (IntN) was fused to various protein cargoes, such as NanoLuc luciferase (Nluc), enhanced green fluorescent protein (eGFP), and Nluc-miniSOG, individually, which led to their successful encapsulation into Encaps to form Cargo@Encap through split intein-mediated protein ligation during protein coexpression and cage assembly in bacteria. Conversely, the SpyCatcher protein was fused to various protein ligands, such as a glutathione binder (GST-SC), dimerizing ligands (FKBP12-SC and FRB-SC), and a cancer-targeting affibody (SC-EGFRAfb); subsequently, they were displayed on Cargo@Encaps through SpyTag/SpyCatcher ligation to form Cargo@Encap/Ligands in a mix-and-match manner. Nluc@Encap/glutathione-S-transferase (GST) was effectively immobilized on glutathione (GSH)-coated solid supports exhibiting repetitive and long-term usage of the encapsulated luciferases. We also established luciferase-embedded layer-by-layer (LbL) nanostructures by alternately depositing Nluc@Encap/FKBP12 and Nluc@Encap/FRB in the presence of rapamycin and applied enhanced green fluorescent protein (eGFP)@Encap/EGFRAfb as a target-specific fluorescent imaging probe to visualize specific cancer cells selectively. Modular functionalization of the interior space and the exterior surface of a protein cage nanoparticle may offer the opportunity to develop new protein-based nanostructured devices and nanomedical tools.

Accessibility-dependent topology studies of membrane proteins using a SpyTag/SpyCatcher protein-ligation system.

Covalent protein-ligation methods were used not only to visualize the localization of proteins of interest in cells, but also to study the topology of plasma and subcellular organelle membrane proteins using fluorescent cell imaging. A 13-amino-acid SpyTag (ST) peptide was genetically introduced either into a variety of subcellular proteins of interest or into different positions of plasma or subcellular organelle membrane proteins individually. Conversely, a 15 kDa SpyCatcher (SC) protein was chemically conjugated with either fluorescent dyes or horseradish peroxidase (HRP) via a thiol-maleimide reaction. The extracellular ST-fused plasma membrane proteins were efficiently labeled with the fluorescent-dye-conjugated SC in both live and permeabilized cells, whereas the intracellularly localized ST-fused subcellular proteins were only labeled in permeabilized cells because of the limited accessibility of the fluorescent-dye-conjugated SC to the membrane. The fluorescent-dye-labeled SC together with selective membrane-permeabilizing agents successfully labeled the plasma or the subcellular organelle membrane proteins in a topology-dependent manner. Moreover, the HRP-conjugated SC not only successfully labeled the ST-fused plasma membrane proteins, thus significantly enhancing fluorescent signals in combination with the tyramide signal amplification agents, but also ligated with an external ST-fused target ligand, thus selectively binding to the endogenously expressed cellular receptors of the target cancer cells.

Development of Recombinant Immunoglobulin G-Binding Luciferase-Based Signal Amplifiers in Immunoassays.

In general immunoassays, secondary antibodies are covalently linked with enzymes and bind to the Fc region of target-bound primary antibodies to amplify signals of low-abundant target molecules. The antibodies themselves are obtained from large mammals and are further modified with enzymes. In this study, we developed novel recombinant immunoglobulin G (IgG)-binding luciferase-based signal amplifiers (rILSAs) by genetically fusing luciferase (Nluc) with antimouse IgG1 nanobody (MG1Nb) and antibody-binding domain (ABD), individually or together, in a mix-and-match manner. We obtained three different highly pure rILSAs in large quantities using a bacterial overexpression system and one-step purification. Mouse-specific rILSA, MG1Nb-Nluc, and rabbit-specific rILSA, Nluc-ABD, selectively bound to target-molecule-bound mouse IgG1 and rabbit IgG primary antibodies, whereas the bispecific rILSA, MG1Nb-Nluc-ABD, mutually bound to both mouse IgG1 and rabbit IgG primary antibodies. All rILSAs exhibited an outstanding signal-amplifying capability comparable to those of conventional horseradish-peroxidase-conjugated secondary antibodies, regardless of the target molecules, in various immunoassay formats, such as enzyme-linked immunosorbent assay, Western blot, and lateral flow assays. Each rILSA was selected for its own individual purpose and applied to various types of target analytes, in combination with a variety of target-specific primary antibodies, effectively minimizing the use of animals as well as reducing the costs and time associated with the production and chemical conjugation of signal-amplifying enzymes.

Development of target-tunable P22 VLP-based delivery nanoplatforms using bacterial superglue.

Protein cage nanoparticles are widely used as targeted delivery nanoplatforms, because they have well-defined symmetric architectures, high biocompatibility, and enough plasticity to be modified to produce a range of different functionalities. Targeting peptides and ligands are often incorporated on the surface of protein cage nanoparticles. In this research, we adopted the SpyTag/SpyCatcher protein ligation system to covalently display target-specific affibody molecules on the exterior surface of bacteriophage P22 virus-like particles (VLP) and evaluated their modularity and efficacy of targeted delivery. We genetically introduced the 13 amino acid SpyTag peptide into the C-terminus of the P22 capsid protein to construct a target-tunable nanoplatform. We constructed two different SpyCatcher-fused affibody molecules as targeting ligands, SC-EGFRAfb and SC-HER2Afb, which selectively bind to EGFR and HER2 surface markers, respectively. We produced target-specific P22 VLP-based delivery nanoplatforms for the target cell lines by selectively combining SpyTagged P22 VLP and SC-fused affibody molecules. We confirmed its target-switchable modularity through cell imaging and verified the target-specific drug delivery efficacy of the affibody molecules displaying P22 VLP using cell viability assays. The P22 VLP-based delivery nanoplatforms can be used as multifunctional delivery vehicles by ligating other functional proteins, as well as affibody molecules. The interior cavity of P22 VLP can be also used to load cargoes like enzymes and therapeutic proteins. We anticipate that the nanoplatforms will provide new opportunities for developing target-specific functional protein delivery systems.

Engineering Tunable Dual Functional Protein Cage Nanoparticles Using Bacterial Superglue.

The selective detection of specific cells of interest and their effective visualization is important but challenging, and fluorescent cell imaging with target-specific probes is commonly used to visualize cell morphology and components and to track cellular processes. Multiple displays of two or more targeting ligands on a polyvalent single template would make it possible to construct versatile multiplex fluorescent cell imaging probes that can visualize two or more target cells individually without the need for a set of individual probes. To achieve this goal, we used encapsulin, a new class of protein cage nanoparticles, as a template and implanted dual targeting capability by presenting two different affibody molecules on a single encapsulin protein cage nanoparticle post-translationally. Encapsulin was self-assembled from 60 identical subunits to form a hollow and symmetric spherical structure with a uniform size. We genetically inserted SpyTag peptides onto the encapsulin surface and prepared various SpyCatcher-fused proteins, such as fluorescent proteins and targeting affibody molecules. We successfully displayed fluorescent proteins and affibody molecules together on a single encapsulin in a mix-and-match manner post-translationally using bacterial superglue, the SpyTag/SpyCatcher ligation system, and demonstrated that these dual functional encapsulins can be used as target-specific fluorescent cell imaging probes. Dual targeting protein cage nanoparticles were further constructed by ligating two different affibody molecules onto the encapsulin surface with fluorescent dyes, and they effectively recognized and bound to two individual targeting cells independently, which could be visualized by selective colors on demand.

Plug-and-playable fluorescent cell imaging modular toolkits using the bacterial superglue, SpyTag/SpyCatcher.

Simple plug-and-playable fluorescent cell imaging modular toolkits are established using the bacterial superglue SpyTag/SpyCatcher protein ligation system. A variety of affibody-fluorescent protein conjugates (AFPCs) are post-translationally generated via the isopeptide bond formation, and each AFPC effectively recognizes and binds to its targeting cells, visualizing them with selective colors on demand.

An enhanced ascorbate peroxidase 2/antibody-binding domain fusion protein (APEX2-ABD) as a recombinant target-specific signal amplifier.

A recombinant target-specific signal amplifier was constructed by genetically fusing the enhanced ascorbate peroxidase 2 (APEX2) and an antibody-binding domain (ABD). The fusion protein APEX2-ABD possessed the peroxidase activity and the antibody-binding capability simultaneously and replaced the conventional HRP-conjugated secondary antibodies in a TSA assay for amplifying fluorescence signals.