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  • Title: Expression, purification, and functional characterization of recombinant PTD-SARA.
    Author: Huang C, Du R, Zhang P, Meng H, Jia H, Song Y, Li M, Zhang Y, Sun S.
    Journal: Acta Biochim Biophys Sin (Shanghai); 2011 Feb; 43(2):110-7. PubMed ID: 21266541.
    Abstract:
    The Smad anchor for receptor activation (SARA) protein is a binding partner for Smad2/3 that plays an important role in the fibrotic promoting signaling pathway initiated by transforming growth factor-β1 (TGF-β1). The C-terminal 665-750 aa of SARA comprises the Smad-binding domain (SBD). By direct interaction through the SBD, SARA inhibits Smad2/3 phosphorylation and blocks the interaction between Smad2/3 and Smad4, thereby restrains the process of fibrosis. In this study, we constructed a SARA peptide aptamer based on the SBD sequence. The recombinant SARA aptamer, fused with a protein transduction domain (PTD-SARA), was cloned, purified from E. coli, and characterized for the first time. The full-length PTD-SARA coding sequence, created with E. coli favored codons, was cloned into a pQE-30 vector, and the recombinant plasmid was transformed into an M15 strain. After Isopropyl β-D-1-Thiogalactopyranoside (IPTG) induction and Ni(2+) affinity purification, recombinant PTD-SARA was further identified by immunoblotting and protein N-terminal sequencing. Epifluorescence microscopy revealed that the recombinant PTD-SARA was transferred into the cytoplasm and nucleus more efficiently than SARA. Moreover, the recombinant PTD-SARA was found to up-regulate the level of E-cadherin and down-regulate the levels of α-SMA and phospho-Smad3 more efficiently than SARA (P < 0.05). Our work explored a method to obtain recombinant PTD-SARA protein. The recombinant PTD-SARA fusion protein could enter HK2 cells (an immortalized proximal tubule epithelial cell line) more efficiently than the SARA protein and reverse the renal epithelial-to-mesenchymal transdifferentiation process that was induced by TGF-β1 more effectively than the SARA protein. Recombinant PDT-SARA is likely to be a potential candidate for clinical prevention and treatment of renal fibrosis.
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