Back to Search Start Over

Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia.

Authors :
Shi P
Wei J
You H
Chen S
Tan F
Lu Z
Source :
Scientific reports [Sci Rep] 2023 Mar 27; Vol. 13 (1), pp. 4943. Date of Electronic Publication: 2023 Mar 27.
Publication Year :
2023

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.<br /> (© 2023. The Author(s).)

Details

Language :
English
ISSN :
2045-2322
Volume :
13
Issue :
1
Database :
MEDLINE
Journal :
Scientific reports
Publication Type :
Academic Journal
Accession number :
36973525
Full Text :
https://doi.org/10.1038/s41598-023-32303-2