title: High-level expression and characterization of bioactive human truncated variant of hepatocyte growth factor in Escherichia coli.
abstract: 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.
authors.affiliation: Heilongjiang Key Laboratory of Tissue Damage and Repair, Mudanjiang Medical University, Mudanjiang, 157011, People's Republic of China.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Wang
authors.forename: Haifeng
authors.initials: H
authors.lastname: Liu
authors.forename: Zhongmin
authors.initials: Z
authors.lastname: Zhang
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.forename: Yuting
authors.initials: Y
authors.lastname: Li
authors.forename: Jinqiu
authors.initials: J
authors.lastname: Gui
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Chu
mesh_terms: D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000502:physiology*; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000199:Actins; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor