Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia
Abstract Hirudin is a pharmacologically active substance in leeches with potent blood anticoagulation properties. Although recombinant hirudin production isolated from Hirudo medicinalis Linnaeus and Hirudinaria manillensis Lesson is known, to our knowledge, this study is the first to report recombi...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-03-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-32303-2 |
| _version_ | 1797853882816135168 |
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| author | Ping Shi Jian Wei Huajian You Shijiang Chen Fayin Tan Zenghui Lu |
| author_facet | Ping Shi Jian Wei Huajian You Shijiang Chen Fayin Tan Zenghui Lu |
| author_sort | Ping Shi |
| collection | DOAJ |
| description | Abstract Hirudin is a pharmacologically active substance in leeches with potent blood anticoagulation properties. Although recombinant hirudin production isolated from Hirudo medicinalis Linnaeus and Hirudinaria manillensis Lesson is known, to our knowledge, this study is the first to report recombinant hirudin expression and production from Hirudo nipponia Whitman. Thus, the present study aimed to clone and characterize the full-length cDNA of a candidate hirudin gene (c16237_g1), which is localized on the salivary gland transcriptome of H. nipponia, and further evaluate its recombinant production using a eukaryotic expression system. The 489-bp cDNA possessed several properties of the hirudin “core” motifs associated with binding to the thrombin catalytic pocket. A fusion expression vector (pPIC9K-hirudin) was constructed and successfully transformed into Pichia pastoris strain GS115 via electroporation. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blot analysis confirmed hirudin expression. The recombinant protein was expressed with a yield of 6.68 mg/L culture. Mass spectrometry analysis further confirmed target protein expression. The concentration and antithrombin activity of purified hirudin were 1.67 mg/mL and 14,000 ATU/mL, respectively. These findings provide a basis for further elucidating the molecular anticoagulation mechanism of hirudin, and address China’s growing market demand for engineered H. nipponia-derived hirudin and hirudin-based drugs. |
| first_indexed | 2024-04-09T19:56:56Z |
| format | Article |
| id | doaj.art-dc74416d4bd4496688858ca2d7a76606 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-09T19:56:56Z |
| publishDate | 2023-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-dc74416d4bd4496688858ca2d7a766062023-04-03T05:25:23ZengNature PortfolioScientific Reports2045-23222023-03-0113111110.1038/s41598-023-32303-2Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponiaPing Shi0Jian Wei1Huajian You2Shijiang Chen3Fayin Tan4Zenghui Lu5Institute of Chinese Caterpillar Fungus, Chongqing Academy of Chinese Materia MedicaDepartment of TCM Geriatrics, Pucheng County HospitalInstitute of Chinese Caterpillar Fungus, Chongqing Academy of Chinese Materia MedicaInstitute of Chinese Caterpillar Fungus, Chongqing Academy of Chinese Materia MedicaInstitute of Chinese Caterpillar Fungus, Chongqing Academy of Chinese Materia MedicaInstitute of Chinese Caterpillar Fungus, Chongqing Academy of Chinese Materia MedicaAbstract Hirudin is a pharmacologically active substance in leeches with potent blood anticoagulation properties. Although recombinant hirudin production isolated from Hirudo medicinalis Linnaeus and Hirudinaria manillensis Lesson is known, to our knowledge, this study is the first to report recombinant hirudin expression and production from Hirudo nipponia Whitman. Thus, the present study aimed to clone and characterize the full-length cDNA of a candidate hirudin gene (c16237_g1), which is localized on the salivary gland transcriptome of H. nipponia, and further evaluate its recombinant production using a eukaryotic expression system. The 489-bp cDNA possessed several properties of the hirudin “core” motifs associated with binding to the thrombin catalytic pocket. A fusion expression vector (pPIC9K-hirudin) was constructed and successfully transformed into Pichia pastoris strain GS115 via electroporation. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blot analysis confirmed hirudin expression. The recombinant protein was expressed with a yield of 6.68 mg/L culture. Mass spectrometry analysis further confirmed target protein expression. The concentration and antithrombin activity of purified hirudin were 1.67 mg/mL and 14,000 ATU/mL, respectively. These findings provide a basis for further elucidating the molecular anticoagulation mechanism of hirudin, and address China’s growing market demand for engineered H. nipponia-derived hirudin and hirudin-based drugs.https://doi.org/10.1038/s41598-023-32303-2 |
| spellingShingle | Ping Shi Jian Wei Huajian You Shijiang Chen Fayin Tan Zenghui Lu Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia Scientific Reports |
| title | Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia |
| title_full | Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia |
| title_fullStr | Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia |
| title_full_unstemmed | Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia |
| title_short | Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia |
| title_sort | cloning characterization and heterologous expression of a candidate hirudin gene from the salivary gland transcriptome of hirudo nipponia |
| url | https://doi.org/10.1038/s41598-023-32303-2 |
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