title: Soluble Expression of a Human MnSOD and Hirudin Fusion Protein inEscherichia coli, and Its Effects on Metastasis and Invasion of 95-D Cells.
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 hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred intoEscherichia colistrain 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.
authors.affiliation: Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China.
authors.forename: Shanze
authors.initials: S
authors.lastname: Yi
authors.affiliation: Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China.
authors.forename: Dewei
authors.initials: D
authors.lastname: Niu
authors.affiliation: School of Life Sciences, Shenzhen University, Shenzhen 518060, P.R. China.
authors.forename: Fang
authors.initials: F
authors.lastname: Bai
authors.affiliation: Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China.
authors.forename: Shuaiguang
authors.initials: S
authors.lastname: Li
authors.affiliation: Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, P.R. China.
authors.forename: Luyuan
authors.initials: L
authors.lastname: Huang
authors.affiliation: Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China.
authors.forename: Wenyan
authors.initials: W
authors.lastname: He
authors.affiliation: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Anand
authors.initials: A
authors.lastname: Prasad
authors.affiliation: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Czachor
authors.affiliation: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Lee Charles
authors.initials: LC
authors.lastname: Tan
authors.affiliation: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Narasaiah
authors.initials: N
authors.lastname: Kolliputi
authors.affiliation: Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China.|Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
mesh_terms: D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006629:Hirudins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D006629:Hirudins; D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase
keywords: DNA protection; hMnSOD-Hirudin; invasion; metastasis; purification; soluble expression