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

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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.


Intracellular uptake of PTD-Hsp27 fusion proteins. Cells were incubated with cy5 conjugated PTD-Hsp27 fusion proteins for 24 h. CLSM images were merged with PTD-conjugated fusion proteins following counterstaining with DAPI
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Fig4: Intracellular uptake of PTD-Hsp27 fusion proteins. Cells were incubated with cy5 conjugated PTD-Hsp27 fusion proteins for 24 h. CLSM images were merged with PTD-conjugated fusion proteins following counterstaining with DAPI

Mentions: PTD-Hsp27 fusion proteins were conjugated by Cy5, following counter-staining with DAPI. As shown in Fig. 4a, florescence of Cy5 in T-Hsp27 group was dispersed more widely in cytoplasm than other groups (H, 9RH, H9R, TH9R), in H9c2 cells. In case of 9R-Hsp27, Hsp27-9R and T-Hsp27-9R groups, less Cy5 florescence intensities were detected in cytoplasm. Among 9R-Hsp27 and Hsp27-9R fusion proteins, 9R-Hsp27 showed less florescence. Florescence intensities were quantified by Image J software.Fig. 4


Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations
Intracellular uptake of PTD-Hsp27 fusion proteins. Cells were incubated with cy5 conjugated PTD-Hsp27 fusion proteins for 24 h. CLSM images were merged with PTD-conjugated fusion proteins following counterstaining with DAPI
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Intracellular uptake of PTD-Hsp27 fusion proteins. Cells were incubated with cy5 conjugated PTD-Hsp27 fusion proteins for 24 h. CLSM images were merged with PTD-conjugated fusion proteins following counterstaining with DAPI
Mentions: PTD-Hsp27 fusion proteins were conjugated by Cy5, following counter-staining with DAPI. As shown in Fig. 4a, florescence of Cy5 in T-Hsp27 group was dispersed more widely in cytoplasm than other groups (H, 9RH, H9R, TH9R), in H9c2 cells. In case of 9R-Hsp27, Hsp27-9R and T-Hsp27-9R groups, less Cy5 florescence intensities were detected in cytoplasm. Among 9R-Hsp27 and Hsp27-9R fusion proteins, 9R-Hsp27 showed less florescence. Florescence intensities were quantified by Image J software.Fig. 4

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.