Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: purification and characterization of the recombinant enzyme.
A glycosylation variant of human tissue-type plasminogen activator (tPA) designated longer-acting tissue-type plasminogen activator (LAtPA) was extensively purified from the milk of a transgenic goat by a combination of acid fractionation, hydrophobic interaction chromatography and immunoaffinity chromatography. This scheme provided greater than 8,000-fold purification of the protein, a cumulative yield of 25% and purity greater than 98% as judged by SDS gel electrophoresis. SDS gel electrophoresis revealed that the transgenic enzyme was predominantly the "two chain" form of the protease. The specific activity of the purified transgenic protein, based on the average of the values obtained for three different preparations, was 610,000 U/mg as judged by amidolytic activity assay. This was approximately 84% of the value observed for the recombinant enzyme produced in mouse C127 cells. Analysis of the transgenic protein indicated that it had a significantly different carbohydrate composition from the recombinant enzyme produced in C127 cells. Molecular size analysis of the oligosaccharides from the transgenic and C127 cell-derived LAtPA preparations confirmed their differences and showed that the mouse cell-derived preparation contained larger, complex-type N-linked oligosaccharide structures than the material produced in goat mammary tissue.
Genzyme Corporation, Framingham, MA 01701.
J
J
Denman
M
M
Hayes
C
C
O'Day
T
T
Edmunds
C
C
Bartlett
S
S
Hirani
K M
KM
Ebert
K
K
Gordon
J M
JM
McPherson
D000818:Animals; D030801:Animals, Genetically Modified; D002241:Carbohydrates / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D014644:Genetic Variation; D006041:Goats / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008892:Milk / Q000201:enzymology*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis; D011247:Pregnancy; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D002241:Carbohydrates; D009844:Oligosaccharides; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator