The aim of the thesis is to develop 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. A variety of target-specific labeling probes have shown that they can be used in fluorescent cell imaging and immunoassays by combining fluorescent molecules or signal-generating enzymes with targeting ligands, including affibody molecules or nanobodies.
Introduction
- Visualization of Target Biomolecules
- Quantitation of Target Biomolecules
- Functional Proteins-based Nanoplatforms
- Protein cage nanoparticles
- Monomeric proteins
- Genetic Encoded Protein Tags
- Research Outline
- Objective for the Thesis
- Outline of the Thesis
The primary antibody binds to the target protein itself or to its epitope, followed by binding to fluorescent dye or enzyme-conjugated secondary antibodies, which respectively emit light at a specific wavelength or react with the substrate to produce colored product (Figure 1.3A) . The phenolic group of the tyramide is catalyzed with hydrogen peroxide and generates reactive tyramide radicals, which covalently bind to nearby tyrosine residues (Figure 1.3B). With this flexibility of SpyTag/SpyCathcer, SpyTag and SpyCatcher can be genetically fused to any type of protein and can be used in vitro and in vivo (Figure 1.7B).
Target-specific Fluorescent Probes for Fluorescence Cell Imaging based on Protein
- Introduction
- Materials and Methods
- Results and Discussion
- Conclusion
The obtained molar concentrations converted to approximately 98% Encap subunits labeled with fluorescence (Figure 2.3), indicating that almost every Encap-L-ST subunit was labeled with fluorescence. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyzes of those reactions showed that the ligation between SC-Afb and fEncap-L-ST took nearly 6 h to complete (Figure 2.4), whereas that of monomeric SC- fused and ST-fused proteins were almost completed within 5 min in previous reports.70,. Each ligated complex bound efficiently to their target cells (Figure 2.5, middle panels), whereas fEncap-L-ST without affibody molecules did not bind to any cell lines (Figure 2.5, top panels).
fEncap ligated with the Affibody molecule also did not bind to MCF-10A or MCF-7 cells, which do not overexpress HER2 and EGFR on their surfaces, respectively (Figure 2.5, bottom panels), suggesting that fEncap-L-ST itself and the affibody molecule ligated. fEncap does not bind significantly to non-targeted cells. TEM images of negatively stained SC protein-ligated Encap confirmed their intact cage architecture and spotted additional densities on the outside, indicating that SC proteins were well displayed on the Encap surface (Figure 2.6c). To avoid this aggregation problem but maximize functionality, we used the 2:2:6 reaction results (Figure 2.8) for further investigation.
Similarly, bifunctional Encaps with SC-EGFRAfb (Encap:mApple:EGFRAfb and Encap:eYFP:EGFRAfb) also selectively bound to their target, MDA-MB-468 cells, with similar fluorescent colors (Figure 2.9), while Encap without affibodies bound not to target cancer cells, which was consistent with previous results (Figure 2.5). Encap-L-ST was labeled with Alexa fluor 546 maleimide, and all Encap-L-ST subunits were also labeled with an Alexa fluor 546 maleimide (Figure 2.10), the same as those in F5M. To check the possibility of cross-targeting of fEncap:HER2Afb and aEncap:EGFRAfb, we treated SK-BR-3 and MDA-MB-468 cells with fEncap:HER2Afb and aEncap:EGFRAfb, respectively (Figures 2.12a and 2.12b), or aEncap :EGFRAfb and fEncap:HER2Afb (Figures 2.12c and 2.12d, respectively).
As expected, green fluorescence only appeared in fEncap:HER2Afb-treated SK-BR-3 cells targeted by HER2Afb (Figure 2.12a), and red only appeared in aEncap:EGFRAfb-treated MDA-MB-468 cells , which is targeted by EGFRAfb. (Figure 2.12b). For multiplex cell imaging, we treated fluorescently labeled Encap-L-ST with two different SC-Afbs (SC-HER2Afb and SC-EGFRAfb) to visualize two or more target cells (Figure 2.11b). Each target cell was detected by the color corresponding to the labeled dye (Figure 2.12e and 2.12f) compared to the negative control cell line, HEK293T (Figure 2.13).
Target-specific Signal Amplifiers for Immunoassays
Introduction
Although enzyme-conjugated secondary antibodies are widely used for signal amplification, their production requires live animals or mammalian cell culture systems with high manufacturing costs, long preparation time, and animal welfare and ethical issues compared to recombinant protein production with bacterial overexpression systems. We attempted to develop IgG-binding recombinant signal enhancers, we have previously introduced antibody-binding domain (ABD) derived from Staphylococcus aureus protein A as a universal non-immunoglobulin IgG binder that has a high binding affinity to and specificity for the Fc region of various IgGs .38, 81 For signal amplification, we fused ABD to glutathione-S-transferase (GST) to create a scaffold protein for conjugation to multiple activated HRPs. HRP-GST-ABD bound to the Fc region of target-bound primary antibodies derived from different species and amplified target-specific signals in both enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry.
However, HRP-GST-ABD lacked species selectivity as it bound to the Fc region of various IgGs regardless of the origin of the primary antibody.73 In a similar approach, Jeong et al. Repebody has been further labeled with various signal generators such as HRP, fluorescent dye, and quantum dots for use in immunoassays and imaging as an alternative to conventional secondary antibodies.82 Meanwhile, single-domain antibodies, also known as nanobodies, are widely used as scaffold proteins. to screen high-affinity binders against specific target molecules for diagnostic and therapeutic purposes Nanobodies derived from camelids or sharks are very stable in a wide temperature range and are easy to use as recombinant fusion proteins with different functional proteins due to their small size and high stability in bacterial overexpression systems. Large quantities of these nanoparticles have been produced in bacterial overexpression systems and directly labeled with fluorophores or enzymes to enhance the signal in various immunoassays and immunostaining.49.
In this study, we prepared two species-specific IgG-binding nanobodies specific for Fc regions of mouse IgG1 and rabbit IgG, designated MG1Nb and RNb, respectively. To establish a plug-and-play modular IgG-binding signal amplifier, we used the well-established SpyTag/SpyCatcher protein ligation system, which allowed us to covalently ligate post-translationally above two individual functional modules. HRP-conjugated SpyCatcher was efficiently ligated to each SpyTag-fused IgG-binding nanobody in a plug-and-play manner.
Target-specific signal amplification was evaluated in Western blot, ELISA, and multiplex TSA cell and tissue imaging (Figure 39 3.1).
Materials and Methods
After three washes in TBST, membranes were incubated for 1 h at room temperature with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (Jackson ImmunoResearch Inc.) or HRP:Nbs (HRP:MG1Nb or HRP:RNb). The blocking buffer (5% goat serum in PBST) was loaded and incubated with gentle shaking for 4 hours at room temperature. After blocking, each well was washed four times with PBST and the solutions of diluted primary antibodies against FLAG (anti-FLAG mouse IgG1 (Abcam) and anti-FLAG rabbit IgG (Thermo-Fisher)) and EpCAM (anti-EpCAM mouse IgG1 and anti- EpCAM rabbit IgG (Sino Biological Inc.)) was loaded into the wells of the target analyte (FLAG-HaloTag protein or EpCAM)-immobilized plates and incubated with gentle shaking for 1 hour at room temperature.
The reaction solutions were incubated for 2 min at room temperature and 2 N H2SO4 solution was immediately added to each well to stop the reaction. Fixed NIH3T6.7 and A431 cells were treated with diluted mouse IgG1 against HER2 and rabbit IgG against EGFR for 2 hours at room temperature. Negative MCF-7 cells were treated in parallel with diluted anti-HER2 mouse IgG1 or anti-EGFR rabbit IgG for 2 h at RT.
For fluorescence cell imaging, Alexa647:MG1Nb and F5M:RNb were incubated with NIH3T6.7 cells and A431 cells, respectively, for 2 h at RT after fixed cell was washed three times with wash buffer. For the TSA assays, the anti-mouse HRP-conjugated secondary antibodies, HRP:MG1Nb, or HRP:RNb were incubated with NIH3T6.7 cells preincubated with anti-HER2 mouse IgG1 for 2 h at RT. The anti-rabbit HRP-conjugated secondary antibodies, HRP:MG1Nb or HRP:RNb were incubated with A431 cells pretreated with anti-EGFR rabbit IgG for 2 h at RT.
After washing three times, the diluted HRP-conjugated secondary antibodies (Sigma-Aldrich) or HRP:Nbs were incubated for 2 h at room temperature.
Results and Discussion
Anti-mouse HRP (Figure 3.6A) and HRP:MG1Nb (Figure 3.6B) secondary antibody probed with mouse IgG1 against FLAG produced similar series of concentration-dependent signals for purified FLAG-HaloTag without significant background noise (Figure 3.6A). . and 3.6B). Almost identical results were also obtained with a secondary antibody conjugated to rabbit HRP (Figure 3.6C) and HRP:RNb (Figure 3.6D) when FLAG-HaloTag was probed with rabbit IgG against FLAG (Figure 3.6C and 3.6D). . Both HRP:MG1Nb and HRP:RNb showed patterns of linear signal enhancement in correlation with sample concentrations and generated signal intensities at each concentration that were very similar to those of conventional HRP-conjugated secondary antibodies (Figures 3.7A and 3.7B).
Both HRP-conjugated anti-mouse (Figure 3.6E) and HRP:MG1Nb (Figure 3.6F) secondary antibodies probed with anti-FLAG mouse IgG1 successfully amplified the concentration-dependent signal series of cell lysates at the expected sites. no significant background (Figure 3.6E and 3.6F). HRP:MG1Nb and HRP:RNb exhibited linear signal amplification patterns depending on the concentration of the target molecule in a complex cell lysate, and the HRP:Nb patterns corresponded well to those of the HRP-conjugated secondary antibodies (Figure 3.7C and 3.7D ). When we applied mouse anti-FLAG IgG1 with anti-mouse HRP-conjugated secondary antibody and HRP:MG1Nb as signal enhancer, we obtained similar representative sigmoidal curves for each HRP construct between the linear absorbance range of 2 to 125 nM for FLAG-HaloTag . proteins (Figure 3.8A).
We also obtained comparable sigmoidal curves between the linear absorbance range of 0.488 to 125 nM using anti-rabbit HRP-conjugated secondary antibody and HRP:RNb in combination with anti-FLAG rabbit IgG (Figure 3.8B). When anti-mouse HRP-conjugated secondary antibody and HRP:MG1Nb were selectively bound to target NIH3T6.7 cells via anti-HER2 mouse IgG1, fluorescence microscopy images revealed extremely high red fluorescence signals (Figure 3.10A, respectively the second and third panels). Using HRP:RNb instead of HRP:MG1Nb generated no fluorescence signal (Figure 3.10A, lower panel).
Fluorescence signal intensity plots showed that TSA with HRP-conjugated secondary antibody or HRP:Nbs generated 5 times more fluorescent signal than that of fluorescently labeled antibodies (Figure 3.10C and 3.10D). To demonstrate simultaneous detections of two different target molecules in the same sample with HRP:MG1Nb and HRP:RNb, we labeled two different targets (skeletal muscle fibers and α-tubulin) in the same whole embryo sample (Figure 3.13C and 3.13D) . We then performed a TSA assay with HRP-conjugated secondary antibodies (Figure 3.13C) or HRP:Nbs (Figure 3.13D), which resulted in target-specific dual-color labeling.
Target-specific Fluorescent Probes for Fluorescent Cell Imaging based on Protein
