Expression of full-length human pro-urokinase in mammary glands of transgenic mice.
Human pro-urokinase expressed in the mammary glands of transgenic animals is quickly activated and converted to urokinase by proteases that are present in the milk. Thus, it is nearly impossible to isolate full-sized pro-urokinase from the milk of transgenic animals. To solve this problem, we constructed transgenic mice that express human pro-urokinase and modified ecotin, which is a potent serine protease inhibitor from E. coli, in their mammary glands. The gene encoding ecotin was modified so as to enhance its specificity for the human urokinase-type plasminogen activator. Co-expression of modified ecotin and human pro-urokinase in the mammary glands allows for purification of full-length human pro-urokinase from these transgenic mice. The results described here suggest a general way of preventing the activation of zymogens that are expressed in the mammary glands of transgenic animals by co-expression of a zymogen along with a protease inhibitor.
Gursky
Yaroslav
Y
Institute of Gene Biology Russian Academy of Sciences, 119334 Moscow, Russia.
Bibilashvili
Robert
R
Minashkin
Mikchail
M
Krasnov
Alex
A
Deikin
Alex
A
Ermolkevich
Tatyana
T
Popov
Andrey
A
Verbovaya
Lilia
L
Rutkevich
Nicolai
N
Shevelev
Alexsander
A
Georgieva
Sofia
S
Razin
Sergey V
SV
Goldman
Igor
I
Sadchikova
Elena
E
D000818:Animals; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D006801:Humans; D008321:Mammary Glands, Animal / Q000201:enzymology*; D051379:Mice; D008822:Mice, Transgenic; D033903:Periplasmic Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
C039076:Eco protein, E coli; D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase