Expression, purification, and functional characterization of recombinant PTD-SARA

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Smad anchor for receptor activation (SARA) protein is crucial in transforming growth factor-β1 (TGF-β1) signaling, regulating fibrosis.
• The Smad-binding domain (SBD) of SARA inhibits Smad2/3 phosphorylation and Smad4 interaction, thus restraining fibrosis.

Purpose of the Study:

• To construct and characterize a recombinant SARA peptide aptamer fused with a protein transduction domain (PTD-SARA).
• To evaluate the efficacy of PTD-SARA in cellular uptake and reversal of TGF-β1-induced renal epithelial-to-mesenchymal transdifferentiation (RMT).

Main Methods:

• Cloning and purification of recombinant PTD-SARA from E. coli.
• Identification via immunoblotting and N-terminal sequencing.
• Assessment of cellular uptake, E-cadherin, α-SMA, and phospho-Smad3 levels using epifluorescence microscopy and Western blotting.

Main Results:

• Recombinant PTD-SARA demonstrated more efficient cellular and nuclear uptake compared to native SARA.
• PTD-SARA significantly upregulated E-cadherin and downregulated α-SMA and phospho-Smad3 levels.
• PTD-SARA more effectively reversed TGF-β1-induced RMT in HK2 cells than SARA.

Conclusions:

• A method for obtaining recombinant PTD-SARA protein was established.
• Recombinant PTD-SARA exhibits superior cellular penetration and antifibrotic activity compared to SARA.
• PTD-SARA represents a potential therapeutic candidate for preventing and treating renal fibrosis.