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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.

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Related in: MedlinePlus

Flow cytometry analysis of PTD-Hsp27 fusion proteins. H9c2 cells were transfected with alexa488 labelled Hsp27 and PTD-Hsp27 fusion proteins, (T-Hsp27, 9R-Hsp27, Hsp27-9R and T-Hsp27-9R). The fluorescence intensity of Alexa488 was measured after 24 h by FACS analysis
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Fig5: Flow cytometry analysis of PTD-Hsp27 fusion proteins. H9c2 cells were transfected with alexa488 labelled Hsp27 and PTD-Hsp27 fusion proteins, (T-Hsp27, 9R-Hsp27, Hsp27-9R and T-Hsp27-9R). The fluorescence intensity of Alexa488 was measured after 24 h by FACS analysis

Mentions: For further comparison of PTD-Hsp27 fusion protein’s transfection efficiency, flow cytometry was performed and mean fluorescence intensity (MFI) from triplicate for each group was recorded. PTD-Hsp27 fusion proteins were labeled with alexa488 according to manufacturer instructions and excessive dye was removed by dialysis in PBS. Cells were treated with PTD-Hsp27 fusion proteins at 5 μM concentration and incubated at 37 °C for 24 h. After incubation, cells were collected by trypsinization followed by washing and centrifugation. Single cell suspension was prepared in FACS buffer (2 % FBS, 0.02 % sodium azide/PBS). Internalization of PTD-Hsp27 fusion proteins was evaluated by a FACS Canto II Caliber flow cytometer (BD Franklin Lakes, NJ) and data was analyzed by Cell Quest Pro software. Figure 5a represents that T-Hsp27 fusion protein retains 90.4 % transfection efficiency significantly higher than 9R-Hsp27 (43.6 %), Hsp27-9R (29.7 %) and T-Hsp27-9R (53.9 %). This data confirms that Tat provides better transfection efficiency for Hsp27 than 9R. As shown in Fig. 5b), the MFI values were also measured with transfection of Alexa 488-conjugated PTD-Hsp27 fusion proteins. The mean fluorescence intensity of T-Hsp27 was 52.7 and relative values of 9R-Hsp27, Hsp27-9R and T-Hsp27-9R groups were about 26.8, 13.9 and 32.3, respectively.Fig. 5


Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations
Flow cytometry analysis of PTD-Hsp27 fusion proteins. H9c2 cells were transfected with alexa488 labelled Hsp27 and PTD-Hsp27 fusion proteins, (T-Hsp27, 9R-Hsp27, Hsp27-9R and T-Hsp27-9R). The fluorescence intensity of Alexa488 was measured after 24 h by FACS analysis
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Flow cytometry analysis of PTD-Hsp27 fusion proteins. H9c2 cells were transfected with alexa488 labelled Hsp27 and PTD-Hsp27 fusion proteins, (T-Hsp27, 9R-Hsp27, Hsp27-9R and T-Hsp27-9R). The fluorescence intensity of Alexa488 was measured after 24 h by FACS analysis
Mentions: For further comparison of PTD-Hsp27 fusion protein’s transfection efficiency, flow cytometry was performed and mean fluorescence intensity (MFI) from triplicate for each group was recorded. PTD-Hsp27 fusion proteins were labeled with alexa488 according to manufacturer instructions and excessive dye was removed by dialysis in PBS. Cells were treated with PTD-Hsp27 fusion proteins at 5 μM concentration and incubated at 37 °C for 24 h. After incubation, cells were collected by trypsinization followed by washing and centrifugation. Single cell suspension was prepared in FACS buffer (2 % FBS, 0.02 % sodium azide/PBS). Internalization of PTD-Hsp27 fusion proteins was evaluated by a FACS Canto II Caliber flow cytometer (BD Franklin Lakes, NJ) and data was analyzed by Cell Quest Pro software. Figure 5a represents that T-Hsp27 fusion protein retains 90.4 % transfection efficiency significantly higher than 9R-Hsp27 (43.6 %), Hsp27-9R (29.7 %) and T-Hsp27-9R (53.9 %). This data confirms that Tat provides better transfection efficiency for Hsp27 than 9R. As shown in Fig. 5b), the MFI values were also measured with transfection of Alexa 488-conjugated PTD-Hsp27 fusion proteins. The mean fluorescence intensity of T-Hsp27 was 52.7 and relative values of 9R-Hsp27, Hsp27-9R and T-Hsp27-9R groups were about 26.8, 13.9 and 32.3, respectively.Fig. 5

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.


Related in: MedlinePlus