Figure 1.
Schematic diagram of monomeric and heptameric targeting ligands.
(A) The cDNA of heptameric ligand consists of coding regions for a target binding domain, a flexible hinge linker, and a heptamerization domain. A 6×His-tag was introduced on the C-terminus of each molecule. The structure of the monomeric targeting ligand is similar to the heptameric ligand with the exception of the absence of the heptamerization domain. (B) Schematic representation of the monomeric and heptameric ligands;; Affibody (Z domain) structure was obtained from PDB database (PDB ID:2B89).
Table 1.
Amino acid sequences of each component of the heptameric targeting ligands.
Figure 2.
SDS-PAGE analysis of the purified monomeric and heptameric targeting ligands.
The purified heptameric ZEGFR, monomeric ZEGFR, heptameric ZHER2, and monomeric ZHER2ligands were separated on a 10% SDS-PAGE gel. About 5 µg of each protein was applied to each lane.
Figure 3.
Native gel separation of monomeric and heptameric targeting ligands.
The purified monomeric ZEGFR, heptameric ZEGFR, monomeric ZHER2 and heptameric ZHER2 ligands were separated on an 8% native gel. About 5 µg of the purified monomer or 20 µg heptamer was loaded to the appropriate lane.
Figure 4.
Determination of the molecular weights of the heptameric targeting ligands by analytical ultracentrifugation analysis.
Purified heptameric ZEGFR and heptameric ZHER2 ligands were centrifuged at 10,000 g for 20 h. Absorbances at 280 nm were recorded every two hours.
Figure 5.
Heat stability assessment of the monomer and the heptamer by circular dichroism analysis.
(A) Monomeric and heptameric ZEGFR, (B) monomeric and heptameric ZHER2 targeting ligands, and (C) heptameric core itself were prepared in a 10 mM phosphate buffer, pH 7.4. Temperature was increased from 25°C to 94°C. Spectra were recorded at various temperatures. The ellipticity at 220 nm was used for the analysis.
Figure 6.
Analysis of the protease resistance of the monomer and the heptamer by thermolysin.
(A) About 5 µg of monomeric and heptameric ZEGFR and (B) monomeric and heptameric ZHER2 targeting ligands were incubated with 100 ng of thermolysin at different temperatures for 20 min. After incubation, reaction was stopped by adding SDS sample buffer and each reaction mixture was separated on a 10% SDS-PAGE to examine protein degradation.
Figure 7.
Binding dynamics of monomeric and heptameric targeting ligands by BIAcore analysis.
The extracellular domain of (A) EGFR and (B) HER2 receptors were immobilized on the CM5 chip. Different concentrations of monomer or heptamer proteins were injected into the channels. Analyses were performed at room temperature at a flow rate of 20 µl/min.
Table 2.
Binding constants of each monomeric and heptameric targeting ligand using BIAcore analysis.
Figure 8.
Cell-based surface receptor binding properties of the monomer and heptamer.
(A) EGFR-positive A431 cells were grown on coverslips. Different concentration of FITC-labeled monomeric and heptameric ZEGFR ligands was incubated with A431 cells for 30 min at 25°C. (B) HER2-positive SK-OV3 cells were grown on coverslips. FITC-labeled monomeric and heptameric ZHER2 ligands were incubated with SK-OV3 cells for 30 min at 25°C. (C) EGFR-negative Jurkat cells and HER2-low expressing MCF7 cells were grown on coverslips. 100 nM of FITC-labeled monomeric and heptameric ligands were incubated with Jurkat and MCF cells for 30 min at 25°C.
Figure 9.
Cell binding analysis by flow cytometry.
(A) 100 nM FITC-monomeric and heptameric ZEGFR ligands were used for labeling of EGFR positive A431 and negative Jurkat cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control. (B) 100 nM FITC-monomeric and heptameric ZHER2 ligands were used for labeling of HER2 positive SK-OV3 and HER2 low expressing MCF7 cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control.
Figure 10.
Co-localization of EEA1 and heptameric targeting ligands.
(A) Two different concentrations of the FITC-labeled heptameric ZEGFR targeting ligands were incubated with A431 cells for 2 h at 37°C. (B) FITC labeled heptameric ZHER2 targeting ligands at two concentrations were incubated with SK-OV3 cells for 2 h at 37°C. EEA1 proteins were detected by Alexa 555-conjugated secondary antibody. Top left panels: cell nuclei stained with DAPI (blue); Top right panels: FITC labeled heptamer (green); bottom left panels: EEA1 antibody (red); bottom right panels: merged image of the three stainings.
Figure 11.
Analysis of cellular toxicity of heptameric targeting ligands by the MTS assay.
A431 and SK-OV3 cells were incubated with various concentrations from 250 nM to 4 µM of each heptameric targeting ligand for 24 h at 37°C. Cisplatin (4 µM) was used as a positive control. Cells without the targeting ligands or without cisplatin were used as a negative control. Absorbance at 490 nm (A490) was measured.