Expression of recombinant Hirudin in transgenic mice milk driven by the goat beta-casein promoter

• Published in: Biotechnology journal Vol. 3; no. 8; p. 1067
• Main Authors: Yen, Chon-Ho, Yang, Chi-Kai, Chen, I-Chung, Lin, Yin-Shen, Lin, Chih-Sheng, Chu, Sen, Tu, Ching-Fu
• Format: Journal Article
• Language: English
• Published: Germany 01.08.2008
• Subjects: Animals; Caseins - genetics; Female; Goats; Hirudins - genetics; Hirudins - metabolism; Mice; Mice, Inbred ICR; Mice, Transgenic; Milk - metabolism; Promoter Regions, Genetic - genetics; Protein Engineering - methods; Recombinant Proteins - metabolism
• ISSN: 1860-7314
• DOI: 10.1002/biot.200800069

Hirudin, isolated from the leech Hirudo medicinalis, inhibits thrombin directly and several expression systems have been used to produce recombinant Hirudin (rHirudin) for pharmaceutical purposes. A DNA fragment containing the Hirudin coding sequence and goat beta-casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat beta-casein expression vector for mouse transgenesis. Four lines of transgenic mice were successfully developed and one line showed a meaningful anti-thrombin activity of 40,000 anti-thrombin units (ATU)/mL in their milk. In this animal line, Hirudin mRNA was found in samples of uterus and kidney with insignificant anti-thrombin activity (</= 280 ATU/g wet tissue); however, mammary glands showed a higher activity of 780 ATU/g wet tissue. Transgenic mice showed no evident physical abnormality. The purified rHirudin was further analyzed by amino acid analysis and was found to contain a tyrosine O-sulfate residue that is absent in rHirudin expression either through Escherichia coli or yeast host systems. Experimental results demonstrated that the beta-casein-promoted Hirudin transgene could be successfully expressed in a murine model and may be applicable to large mammals such as livestock for mass production of rHirudin for pharmaceuticals.