Expression of recombinant Hirudin in transgenic mice milk driven by the goat beta-casein promoter.
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.
Yen
Chon-Ho
CH
Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan, R.O.C.
Yang
Chi-Kai
CK
Chen
I-Chung
IC
Lin
Yin-Shen
YS
Lin
Chih-Sheng
CS
Chu
Sen
S
Tu
Ching-Fu
CF
D000818:Animals; D002364:Caseins / Q000235:genetics*; D005260:Female; D006041:Goats; D006629:Hirudins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D008892:Milk / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
D002364:Caseins; D006629:Hirudins; D011994:Recombinant Proteins