Development of Functional Protein-based Target-specific Labeling Nanoplatforms for Biological Applications

Department of Biological SciencesQualitative and quantitative analyses of target biomolecules in the cell are essential for the early detection of diseases and the prognosis of treatment in various biomedical research and clinical applications. To selectively detect and quantify biomolecules of interest in complicated biological samples, various methods have been developed in the biotechnology fields. Target biomolecules in the cells can be specifically visualized with labeling probes by fluorescent cell imaging and quantified with immunoassay based on antigen-antibody interactions. The aim of the thesis is to develop the functional protein-based target-specific labeling nanoplatforms for application in fluorescent cell imaging and immunoassays. Target biomolecules within the cell can be detected with target-specific fluorescent cell imaging probes. Protein cage nanoparticles as attractive polyvalent nanoplatforms have been applied to bioimaging probes and biosensor components because they have a well-defined symmetric hollow shell structure with uniform nanoscale particle sizes. Their polyvalent nature allows uniform multiple targeting ligands or fluorescent probes to genetically and/or chemically adhere to their surface. A new class of protein cage nanoparticles, encapsulin, was developed as a tunable dual-functional nanoplatform, which has its target-specific capability with multiple combinations of targeting ligands and colors, using bacterial glue, the SpyTag/SpyCatcher (ST/SC) protein ligation system. Next, target-specific signal amplifiers were developed as secondary antibody mimics to be applied in immunoassays. Immunoassays are utilized to selectively detect and quantify lowabundance biomolecules in biological samples through antigen-antibody interaction. HRPconjugated IgG-binding nanobodies were established using ST/SC protein ligation system. They have selective and strong binding to specific IgG and show signal amplifying capability in various types of immunoassays, such as western blot, ELISA, and the multiplex TSA cell and tissue imaging. A variety of target-specific labeling probes demonstrated that they could be utilized in fluorescent cell imaging and immunoassays by combining fluorescent molecules or signal generating enzyme with targeting ligands, including affibody molecules or nanobodies.ope

Exploiting dual functional cell imaging modular toolkit using bacterial superglue, SpyTag/SpyCatcher

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. Multiple fluorescent cell imaging probes that are simple and target-specific can be generated by combining target-specific affibodies and sensitive fluorescent proteins using bacterial superglue, the SpyTag/SpyCatcher ligation system. Further, 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. 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 mixing-and-matching manner post-translationally using bacterial superglue 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.close

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

Diphtheria toxin-based efficient target-switchable anticancer drug

Recombinant immunotoxins (RITs) have been extensively utilized in the field of targeted cancer therapy. RITs are composed of toxins derived from natural bacterial strains and antibodies for target specific killing of cancer cells. The conjugated antibodies have specificities against the surface receptors of target cancer cells, facilitate the toxin internalization and consequently induce fatal effects to the target cell. However, low intracellular penetration and endosomal escape efficiency has limited the application of RITs. Diphtheria toxin is a candidate toxin which has potential of overcoming those limitation of RIT. The diphtheria toxins are produced by Corynebacterium diphtheriae and belong to AB toxin families which consist of catalytic (A) domain, binding (B) domain, and translocation(T) domain. Once binding domain of the diphtheria toxin bind to the specific target receptors, it facilitates the receptor-mediated endocytosis. Translocation domain delivers the catalytic domain from endosome to cytosol, which helps its catalytic domain to escape endosome. By substituting the binding domain with recombinant affinity molecules and using the diphtheria toxin, it is expected to overcome the low endosomal escape efficiency of the conventional RITs. In this study, we designed the diphtheria toxin-based target-switchable modular anticancer drugs by using protein ligation system, killing target cells specifically. To create module toxin and module affibody, the catalytic and translocation domain of diphtheria toxin (DTA), and cancer specific affibodies were prepared, genetically fused with protein ligation modules. By simple mix of module diphtheria toxin and module affibodies, we can easily introduce the target-specific diphtheria toxin (DTA-Afb) within an hour. Finally, we confirmed the targeted toxicity against the specific cancer in vitro as well as in vivo. DTA-Afb shows much higher toxicity than doxorubicin, which is a general anticancer drug. The diphtheria toxin-based RIT is promising method for targeted anticancer drug