Soluble Expression of a Human MnSOD and Hirudin Fusion Protein in Escherichia coli, and Its Effects on Metastasis and Invasion of 95-D Cells
| Autor: | Feng Wang, Anand Prasad, Alexander Czachor, Lee Charles Tan, Narasaiah Kolliputi, Shuaiguang Li, Dewei Niu, Luyuan Huang, Wenyan He, Shanze Yi, Fang Bai |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification lac operon General Medicine medicine.disease_cause Applied Microbiology and Biotechnology Fusion protein Molecular biology 03 medical and health sciences 030104 developmental biology Enzyme chemistry Affinity chromatography Gentamicin protection assay Cell culture medicine Cytotoxicity Escherichia coli Biotechnology |
| Zdroj: | Journal of Microbiology and Biotechnology. 26:1881-1890 |
| ISSN: | 1738-8872 1017-7825 |
| Popis: | 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 hMnSOD-Hirudin 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. |
| Databáze: | OpenAIRE |
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