The
functional peptide cys-TAT was conjugated on the surface of rQβ@b-3WJ
to enhance cell uptake.23 (link) The Cys-TAT peptides
(KYGRRRQRRKKRG-cys-SH) were conjugated to rQβ@b-3WJ by sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1carboxylate (sulfo-SMCC; Sigma-Aldrich,
St. Louis, MO, USA) as a cross-linker. Briefly, 5 μL of sulfo-SMCC
solution (10 mg/mL in deionized (DI)-H2O) was added to
a 600 μL solution of 2 μM rQβ@b-3WJ in PBS buffer
(pH = 7.4) for 30 min at 25 °C in the dark and then purified
using a filter column (Amicon Ultra15 Centrifugal Filter Units. 100,000
MWCO. Merck Millipore, LOT: R6EA45140, Ireland) with PBS buffer. The
samples were desalted with a filter column (100,000 MWCO) and washed
3 times with PBS buffer. Subsequently, the maleimide-terminated rQβ@b-3WJ
was reacted with 30 μL of Cys-TAT solution (0.3 mg/mL) at 25
°C for 2 h in the dark and then purified again using the above-mentioned
procedure to obtain TrQβ@b-3WJ.
To confirm the successful
conjugation of TAT peptides on Qβ@b-3WJsiLUCsiEGFR. The Qβ@b-3WJsiLUCsiEGFR and TQβ@b-3WJsiLUCsiEGFR were
mixed with 2-mercaptoethanol (SigmaAldrich, St. Louis, MO, USA) and
incubated at 95 °C for disulfide bond breaking and protein denaturing.
The denatured samples were analyzed by SDS-PAGE (12%) electrophoresis
and then stained using Coomassie Brilliant Blue R-250 Dye (Sigma–Aldrich,
St. Louis, MO, USA).
functional peptide cys-TAT was conjugated on the surface of rQβ@b-3WJ
to enhance cell uptake.23 (link) The Cys-TAT peptides
(KYGRRRQRRKKRG-cys-SH) were conjugated to rQβ@b-3WJ by sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1carboxylate (sulfo-SMCC; Sigma-Aldrich,
St. Louis, MO, USA) as a cross-linker. Briefly, 5 μL of sulfo-SMCC
solution (10 mg/mL in deionized (DI)-H2O) was added to
a 600 μL solution of 2 μM rQβ@b-3WJ in PBS buffer
(pH = 7.4) for 30 min at 25 °C in the dark and then purified
using a filter column (Amicon Ultra15 Centrifugal Filter Units. 100,000
MWCO. Merck Millipore, LOT: R6EA45140, Ireland) with PBS buffer. The
samples were desalted with a filter column (100,000 MWCO) and washed
3 times with PBS buffer. Subsequently, the maleimide-terminated rQβ@b-3WJ
was reacted with 30 μL of Cys-TAT solution (0.3 mg/mL) at 25
°C for 2 h in the dark and then purified again using the above-mentioned
procedure to obtain TrQβ@b-3WJ.
To confirm the successful
conjugation of TAT peptides on Qβ@b-3WJsiLUCsiEGFR. The Qβ@b-3WJsiLUCsiEGFR and TQβ@b-3WJsiLUCsiEGFR were
mixed with 2-mercaptoethanol (SigmaAldrich, St. Louis, MO, USA) and
incubated at 95 °C for disulfide bond breaking and protein denaturing.
The denatured samples were analyzed by SDS-PAGE (12%) electrophoresis
and then stained using Coomassie Brilliant Blue R-250 Dye (Sigma–Aldrich,
St. Louis, MO, USA).
