High-level expression and characterization of bioactive human truncated variant of hepatocyte growth factor in Escherichia coli

• Published in: World journal of microbiology & biotechnology Vol. 30; no. 11; pp. 2851 - 2859
• Main Authors: Wang, Xiaohua, Liu, Haifeng, Zhang, Zhongmin, Liu, Yang, Li, Yuting, Gui, Jinqiu, Chu, Yanhui
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.11.2014; Springer Nature B.V
• Subjects: Actins - biosynthesis; Actins - genetics; Analysis; Applied Microbiology; Bacteria; Biochemistry; Biocompatibility; Biological activity; Biomedical and Life Sciences; Biotechnology; Cell Proliferation - drug effects; Cells, Cultured; Cellular; Collagen Type I - biosynthesis; Collagen Type I - genetics; E coli; Environmental Engineering/Biotechnology; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Gene expression; Gene Expression Profiling; Gene Expression Regulation - drug effects; Genes; Growth factors; Hepatic Stellate Cells - drug effects; Hepatic Stellate Cells - physiology; Hepatocyte Growth Factor - biosynthesis; Hepatocyte Growth Factor - genetics; Hepatocyte Growth Factor - isolation & purification; Humans; In vitro testing; Laboratories; Life Sciences; Liver; Microbiology; Original Paper; Protein expression; Proteins; Recombinant; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; RNA, Messenger - analysis; RNA, Messenger - genetics; Smooth muscle; Studies; Hepatic fibrosis; Anti-fibrotic; Non-fusion protein; Hepatocyte growth factor
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-014-1711-3

Hepatocyte growth factor (HGF) is an effective anti-fibrotic factor because of its bioactivity in inhibiting fibrosis-related proteins in the development of hepatic fibrosis. However, high-level production of bioactive mature form HGF is difficult because of its complex structure. Here, we report a non-fusion protein expression system to obtain truncated variant of N-terminal hairpin and first kringle domains of HGF (tvNK1) in Escherichia coli to determine its anti-fibrotic effects on hepatic stellate cells (HSCs). Under the selected conditions of cultivation and isopropyl-β-D-1-thiogalactopyranoside induction, the expression level of tvNK1 accounted for approximately 65 % of the total cellular protein and 50 % of fusion protein in the supernatant of whole cell lysates. The recombinant protein could be purified in one step with Ni 2+ -affinity chromatograph. Finally, about 65 mg recombinant tvNK1 was obtained from 1 l fermentation culture with no <95 % purity. In vitro, the final purified tvNK1 was shown to inhibit the proliferation of HSCs and decrease the mRNA and protein expression levels of fibrosis-related COL1A1 and α-smooth muscle actin genes.