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, 52 (5), 324-329

Enhanced Delivery of Protein Fused to Cell Penetrating Peptides to Mammalian Cells

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Enhanced Delivery of Protein Fused to Cell Penetrating Peptides to Mammalian Cells

Jung-Il Moon et al. BMB Rep.

Abstract

Recent progress in cellular reprogramming technology and lineage-specific cell differentiation has provided great opportunities for translational research. Because virus-based gene delivery is not a practical reprogramming protocol, protein-based reprogramming has been receiving attention as a safe way to generate reprogrammed cells. However, the poor efficiency of the cellular uptake of reprogramming proteins is still a major obstacle. Here, we reported key factors which improve the cellular uptake of these proteins. Purified red fluorescent proteins fused with 9xLysine (dsRED-9K) as a cell penetrating peptide were efficiently delivered into the diverse primary cells. Protein delivery was improved by the addition of amodiaquine. Furthermore, purified dsRED-9K was able to penetrate all cell lineages derived from mouse embryonic stem cells efficiently. Our data may provide important insights into the design of protein-based reprogramming or differentiation protocols [BMB Reports 2019; 52(5): 324-329].

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures

Fig. 1
Fig. 1
(A) Schematic diagram of E. coli expression vectors for dsRED, dsRED-9R, and dsRED-9K. (B) Cell penetrating capacity of dsRED, dsRED-9R, and dsRED-9K in both an immortalized cell line and primary cells which were cultured in 24-well plates and treated with 20 μg/ml of either dsRED-9R or dsRED-9K proteins. After 6 hrs of incubation, fluorescent images were captured with matching exposure to test the efficiency of penetration. Penetrations of both dsRED-9R and dsRED-9K, but not dsRED alone, were observed in all three target cell types. (C) Penetrating efficiency of purified dsRED-9K was compared to that of the extracts prepared from HEK293 cells expressing pCMV dsRED-9K. Human fibroblasts on 12-well plates were treated with purified dsRED-9K and whole cell extracts of HEK293 cells transfected with dsRED-9K construct. After 6 hrs of incubation, fluorescent images were captured. Purified dsRED-9K showed better distribution of signals in the cell body and surface.
Fig. 2
Fig. 2
(A) Penetration time-course of purified dsRED-9K was checked. HEK293 cells were plated in a 12-well plate and treated with 20 μg of dsRED-9K in their culture medium for 24 hrs. Fluorescent images were acquired 8 times in 24 hrs with matching exposure. (B) Images were analyzed by CellProfiler (version 2.1.1; cellprofiler.org) for measuring the fluorescence intensity. Three independent fields from each time point were used with the matching exposure. Each cell was identified by object identification modules, followed by measurement of red fluorescent intensity.
Fig. 3
Fig. 3
Transduction ability of dsRED-9K was enhanced by amodiaquine (AQ). Human fibroblasts (A) and CHO cells (B) were pre-treated with different concentrations of AQ and then treated with 20 μg/ml of dsRED-9K. After 6 hrs of incubation, fluorescent images were captured. AQ further increased the penetrating efficiency of the purified dsRED-9K proteins in a dose-dependent manner. (C) Images were analyzed by CellProfiler.
Fig. 4
Fig. 4
(A) mESCs were differentiated using the neuronal differentiation protocol described in the Materials and Methods section. The formation of typical cell types expected in the course of neuronal differentiation were observed. (B) The penetration of dsRED-9K was checked in mouse ES cells cultured on 24-well plates and confirmed by detection of the emitted red fluorescence. (C) ES cells differentiated to embryoid bodies (EB). 20 μg/ml of dsRED-9K was added to the culture media and the delivery of dsRED-9K was confirmed by detection of the emitted red fluorescence. (D, E) EBs were differentiated to neurons. Neural precursors (D) and neurons (E) were collected at day 16 and day 25, respectively. 20 μg/ml of dsRED-9K was added to the culture media and the delivery of dsRED-9K was confirmed by detection of the emitted red fluorescence. Nestin and Tuj1 correspond to dsRED-9K proteins in neural precursors and neurons, respectively. The dsRED-9K protein can penetrate all the cell types formed during the course of in vitro mESC differentiation.

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