• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.61-64
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• 2001

Delta-integration vectors were constructed for the purpose of achieving homologous integration of the hirudin expression cassette into the chromosome of Saccharomyces cerevisiae. A double $\delta$ system truncated with the unnecessary bacterial genes, and consequently having a reduced insert size for integration, showed a four-fold increase in transformation efficiency at given DNA concentrations, and as a result, the constructed recombinant yeast strain had a 1.3-fold enhancement in hirudin expression level compared with a single $\delta$ system.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). 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 $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, 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.

• Journal of Microbiology and Biotechnology
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• v.3 no.3
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• pp.161-167
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• 1993

Immobilized metal ion affinity chromatography (IMAC) was used to separate various types of recombinant hirudins from the culture broth. The wild type hirudin exhibited a retention in Cu(II)-chelated affinity chromatgoraphy since it contained a single exposed histidine at position 51. To obtain a stronger retention on an IDA-Cu(II) column, the hirudin variants were genetically engineered to contain one or two histidine (s) more than the wild type. While the affinity of the variants for IDA-Cu(II) ligand increased in comparison to that of the wild type, the antithrombin activities reduced to a certain degree. Cu(II), Ni(II) and Zn(II) ions were applied separately to the metal chelate column to investigate ligand specificity with respect to protein retention. As a result, the Cu(II) chelated chromatography gave the best resolution for all the hirudins tested and appeared to be the only IMAC that could be used generally for the purification of hirudins with a decreasing pH gradient.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.391-391
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• 2005