Expression, purification, and functional characterization of recombinant PTD-SARA.
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.
Department of Nephrology, The First Affiliated Hospital, Fourth Military Medical University, Xi'an, China. drchenhuang@163.com
Chen
C
Huang
Rui
R
Du
Peng
P
Zhang
Hua
H
Meng
Huiwei
H
Jia
Yang
Y
Song
Man
M
Li
Yingqi
Y
Zhang
Shiren
S
Sun
D000595:Amino Acid Sequence; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D054338:Cell Transdifferentiation / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D005822:Genetic Vectors / Q000096:biosynthesis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051785:Smad Proteins / Q000235:genetics* / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D015854:Up-Regulation
D015820:Cadherins; D047908:Intracellular Signaling Peptides and Proteins; C557100:PTD-SARA fusion protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; C116420:ZFYVE16 protein, human; D012697:Serine Endopeptidases