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Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations

View Article: PubMed Central - PubMed

ABSTRACT

Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein’s nature, transduction efficiency and stability.

No MeSH data available.


In vitro cytotoxicity of PTD-Hsp27 fusion proteins in H9c2 cells
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Fig3: In vitro cytotoxicity of PTD-Hsp27 fusion proteins in H9c2 cells

Mentions: The cytotoxicity of all PTD-Hsp27 recombinant fusion proteins in H9c2 cells was tested in dose dependent mode from 1 to 10 μM. Cells were treated with 1, 3, 5, 7 and 10 µM PTD-Hsp27 purified fusion proteins for 24 h. MTT assay was used to determine cell viability. As shown in Fig. 3, no significant cytotoxicity was observed in any of the PTD-Hsp27 fusion proteins treated groups within the assigned dose-range of 1–10 μM. For further experiments, the protein concentration was fixed at 5 μM. These results suggest that PTDs such as 9R and Tat could be used as safe therapeutic protein carriers with minimum toxicity in mammalian cells regardless from their site of attachment with therapeutic protein Hsp27.Fig. 3


Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations
In vitro cytotoxicity of PTD-Hsp27 fusion proteins in H9c2 cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037156&req=5

Fig3: In vitro cytotoxicity of PTD-Hsp27 fusion proteins in H9c2 cells
Mentions: The cytotoxicity of all PTD-Hsp27 recombinant fusion proteins in H9c2 cells was tested in dose dependent mode from 1 to 10 μM. Cells were treated with 1, 3, 5, 7 and 10 µM PTD-Hsp27 purified fusion proteins for 24 h. MTT assay was used to determine cell viability. As shown in Fig. 3, no significant cytotoxicity was observed in any of the PTD-Hsp27 fusion proteins treated groups within the assigned dose-range of 1–10 μM. For further experiments, the protein concentration was fixed at 5 μM. These results suggest that PTDs such as 9R and Tat could be used as safe therapeutic protein carriers with minimum toxicity in mammalian cells regardless from their site of attachment with therapeutic protein Hsp27.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein’s nature, transduction efficiency and stability.

No MeSH data available.