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  • br Production of peptides and conjugation

    2020-03-17


    2.2. Production of peptides and conjugation with Qdot or MNPs
    E. coli BL21(DE3) strain bearing plasmid pTH-BirA with either plasmid pET28-TZH2 or plasmid pET-TCoh were grown in Luria-Bertani medium supplemented with 4 μM biotin (Sigma-Aldrich, MO, USA) at 30 °C. The growth of bacteria was measured turbidi-
    metrically at 550 nm (OD550). Upon reaching 0.3 at OD550, recom-binant strains were induced for the peptide production by adding
    50 μM IPTG and continued to grow for 4 h. Bacterial cells were then harvested by centrifugation and re-suspended in phosphate buffer (pH 7.4). Followed by sonication, the cell lysate was recov-ered and used for the purification of peptides by His GraviTrap kit (GE Healthcare, NJ, USA) according to the manufacture’s pro-tocol. The purified peptides were quantified by Bio-Rad Protein Assay (Bio-Rad Laboratiries, CA, USA) and stored in 50% glycerol at −20 °C until use. The removal of TrxA from the peptide was conducted with Enterokinase (New England Biolab Inc., Ontario, Canada) for 16 h at room temperature. Digested peptides were iso-lated by the His GraviTrap kit. Finally, peptides were incubated 
    with either Qdot605-streptavidin (Life Technologies, Grand Island, NY, USA) or SiMAG-streptavidin (Chemicell, Berlin, Germany) in the phosphate-saline buffer (PBS) at Epirubicin HCl 30 °C for 1 h. The conjugation method essentially followed the manufacture’s protocol.
    Human breast cancer cell lines including MCF-7, MDA-MB-231, SKBR3, and MCF-7/her18 cells were kindly provided by Dr. M. C. Kao (China Medical University, Taiwan). Cell lines were maintained in DMEM/F12 medium (HyClone Lab Inc., Logan, UT) supplemented with 10% fetal bovine serum at 37 °C in a humidified Epirubicin HCl of 5% CO2. Cell concentrations were measured by hemocytometer and the cell culture medium was changed every 2 days.
    2.4. Analysis by fluorescence and confocal microscopy
    Cell lines were rinsed with PBS and fixed with 3.7% paraformaldehyde (Sigma-Aldrich). Blocked with 3% BSA, cells were incubated with the peptide (5 nM) for 1 h at room temperature. Cells were repeatedly washing with PBS before the application of anti-TrxA antibody at 1:200 (Santa Cruz Biotech Inc., CA, USA). The reaction proceeded for 1 h and the unbounded antibody was washed out with PBS. Subsequent administration of the anti-rabbit IgG-FITC at 1:500 (Jackson Immuno Research Lab. Inc., PA, USA) was performed for another one hour. After washing with PBS, cells were stained with 1 μg/mL DAPI (Sigma-Aldrich) and Phalloidin-TRITC (Sigma-Aldrich) at 1:500 for the observation of cell nucleus and cytoskeleton by fluorescent microscopy (IX71 Olympus, Tokyo, Japan).
    The experiments with the peptide and anti-ErbB2 A body® (Abcam, Cambridge, UK) were carried out by seeding cells into 24-well plates with coverslips on the day before the assay. In one as-say, cells were rinsed with the serum-free medium and PBS once and then fixed with 3.7% paraformaldehyde. After washing with PBS, cells were incubated with the peptide (5 nM) and stained with 1 μg/mL DAPI for the further analysis. In another assay, living cells were directly incubated with either the peptide or A body® molecule (5 nM) after rinsing with serum-free medium and PBS. At the end of the treatment, cells were fixed with paraformalde-hyde and stained with DAPI. The anti-HER2/neu antibody (9G6) at 1:200 (Santa Cruz Biotech Inc.) was subsequently applied to cells, followed by the administration of the secondary antibody anti-mouse IgG-FITC at 1:500. Cells were washed with PBS and mounted for observation by fluorescent microscopy and confocal microscopy (LCS SP2, Wetzlar, Germany).
    2.5. In vitro hyperthermia
    The non-covalent fusion of two peptides proceeds via the in-teraction of Doc and Coh. This reaction was carried out by incu-bation of the two peptides at an equal molar concentration in PBS containing 20 μM CaCl2 at 30 °C for 1 h. Cancer cells were admin-istrated with the resulting peptide for 5 min, followed by wash-ing cells with PBS. Hyperthermia was performed with the alternat-ing magnetic field (AMF) generator HFI-3 (RDO Induction L.L.C., NJ, USA) at 2.4 kW and 250 kHz. The cell-counting kit (Dojindo Molec-ular Technologies, Inc. MD, USA) was used and the absorbance of cells in each well was measured with a microplate reader at 450 nm (SpectraMax M2, Molecular Device, USA). Cell viability was determined by dividing the absorbance of the treated cells with that of untreated cells.