Isolation and molecular cloning of hepatocyte growth factor from guinea pig (gHGF), and expression of truncated variant of gHGF with improved anti-fibrotic activity in Escherichia coli

Abstract

Hepatocyte growth factor (HGF) is an attractive target for anti-fibrotic therapy because it attenuates excessive transforming growth factor-β1 (TGF-β1) which plays an important role in hepatic fibrosis. In the study, we reported on the isolation and molecular cloning of the open reading frame (ORF) of guinea pig HGF (gHGF), encoding a protein of 729 amino acids, with an apple-like (hairpin) domain, four kringle domains and a trypsin-like serine protease domain. Moreover, the truncated variant of gHGF (a double mutant of N-terminal hairpin and first kringle domains of gHGF, K132E and G134E, gmNK1) protein fused with His₆ tag, the molecular weight of which was about 20.0kDa, which was expressed in Escherichia coli BL21 (DE3) and purified with Ni²⁺-affinity chromatography. Furthermore, gmNK1 inhibited protein expression levels of fibrosis-related type I collagen (Col I) and α-smooth muscle actin (α-SMA) genes in TGF-β1-activated HSC-T6 cells and CCl₄-induced liver fibrosis in rat. In addition, gmNK1 ameliorated liver morphology and fibrotic responses in fibrosis animal. Taken together, we first reported on the sequence of HGF from guinea pig and determined the anti-fibrotic activity of gmNK1 in hepatic fibrosis, which will be helpful for investigations into the biological roles of gHGF in this important animal model.

Keywords: Hepatic fibrosis; Hepatic stellate cells; Hepatocyte growth factor.