|
KMID : 0545120160260111881
|
|
Journal of Microbiology and Biotechnology
2016 Volume.26 No. 11 p.1881 ~ p.1890
|
|
|
Soluble Expression of a Human MnSOD and Hirudin Fusion Protein in Escherichia coli, and Its Effects on Metastasis and Invasion of 95-D Cells
|
|
Yi Shan Ze
Niu De Wei
Bai Fang
Li Shuai Guang
Huang Lu Yuan
He Wen Yan
Prasad Anand
Czachor Alexander
Tan Lee Charles
Kolliputi Narasaiah
Wang Feng
|
|
|
Abstract
|
|
|
|
Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals (O2-). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSODHirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was 2,444.0 ± 96.0 U/mg, and the anticoagulant activity of the hMnSOD-Hirudin protein was 599.0 ± 35.0 ATU/mg. In addition, in vitro bioactivity assay showed that the hMnSODHirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human CD4 +, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.
|
|
|
KEYWORD
|
|
|
hMnSOD-Hirudin, Soluble Expression, Purification, DNA protection, invasion, metastasis
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
  
|
|