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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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PTD-Hsp27 fusion proteins purification and expression. a Schematic structures of vector constructs b identification of PTD-Hsp27 fusion proteins by SDS-PAGE and c western blot. H Heat shock protein 27, TH Tat-Hsp27, 9RH Nona-arginine-Hsp27, H9R Hsp27-Nona-arginine, T-H9R Tat-Hsp27-Nona-arginie
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Fig2: PTD-Hsp27 fusion proteins purification and expression. a Schematic structures of vector constructs b identification of PTD-Hsp27 fusion proteins by SDS-PAGE and c western blot. H Heat shock protein 27, TH Tat-Hsp27, 9RH Nona-arginine-Hsp27, H9R Hsp27-Nona-arginine, T-H9R Tat-Hsp27-Nona-arginie

Mentions: The pET28a vector was used to construct pET28a-Tat and pET28a-9R vectors with C-terminal poly histidine (6× His) affinity tag. Hsp27 genes were cloned into pET28a-Tat and pET28a-9R vectors. Vector designs of PTD-Hsp27 fusion proteins and their nucleotide sequences were confirmed by sequence analysis. Tat and 9R were cloned to C-terminus or N-terminus of Hsp27. Vector constructs of Hsp27 recombinant proteins are shown in Fig. 2a.Fig. 2


Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations
PTD-Hsp27 fusion proteins purification and expression. a Schematic structures of vector constructs b identification of PTD-Hsp27 fusion proteins by SDS-PAGE and c western blot. H Heat shock protein 27, TH Tat-Hsp27, 9RH Nona-arginine-Hsp27, H9R Hsp27-Nona-arginine, T-H9R Tat-Hsp27-Nona-arginie
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: PTD-Hsp27 fusion proteins purification and expression. a Schematic structures of vector constructs b identification of PTD-Hsp27 fusion proteins by SDS-PAGE and c western blot. H Heat shock protein 27, TH Tat-Hsp27, 9RH Nona-arginine-Hsp27, H9R Hsp27-Nona-arginine, T-H9R Tat-Hsp27-Nona-arginie
Mentions: The pET28a vector was used to construct pET28a-Tat and pET28a-9R vectors with C-terminal poly histidine (6× His) affinity tag. Hsp27 genes were cloned into pET28a-Tat and pET28a-9R vectors. Vector designs of PTD-Hsp27 fusion proteins and their nucleotide sequences were confirmed by sequence analysis. Tat and 9R were cloned to C-terminus or N-terminus of Hsp27. Vector constructs of Hsp27 recombinant proteins are shown in Fig. 2a.Fig. 2

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