Expression, purification, and evaluation of in vivo anti-fibrotic activity for soluble truncated TGF-β receptor II as a cleavable His-SUMO fusion protein.

Abstract

Excessive production of transforming growth factor-β1 (TGF-β1) and its binding to transforming growth factor-β receptor type II (TGF-βRII) promotes fibrosis by activation of the TGF-β1-mediated signaling pathway. Thus, the truncated extracellular domain of TGF-βRII (tTβRII) is a promising anti-fibrotic candidate, as it lacks the signal transduction domain. In this work, the native N-terminal tTβRII was prepared as a His-SUMO fusion protein (termed His-SUMO-tTβRII) in Escherichia coli strain BL21 (DE3). His-SUMO-tTβRII was expressed as a soluble protein under optimal conditions (6h of induction with 0.5mM IPTG at 37°C). His-SUMO-tTβRII was purified by Ni-NTA resin chromatography, and then cleaved with SUMO protease to release native tTβRII, which was re-purified using a Ni-NTA column. Approximately 12mg of native tTβRII was obtained from a one liter fermentation culture with no less than 95% purity. In vivo studies demonstrated that tTβRII prevented CCl4-induced liver fibrosis, as evidenced by the inhibition of fibrosis-related Col I and α-SMA protein expression in C57BL/6 mice. In addition, tTβRII downregulated phosphorylation of SMAD2/3, which partly repressed TGF-β1-mediated signaling. These data indicate that the His-SUMO expression system is an efficient approach for preparing native tTβRII that possesses anti-liver fibrotic activity, allowing for the large-scale production of tTβRII, which potentially could serve as an anti-fibrotic candidate for treatment of TGF-β1-related diseases.