Purification and characterization of human plasminogen activator inhibitor type I expressed in Saccharomyces cerevisiae.
The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.
Gardell
S J
SJ
Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Hare
T R
TR
Han
J H
JH
Markus
H Z
HZ
Keech
B J
BJ
Carty
C E
CE
Ellis
R W
RW
Schultz
L D
LD
D000941:Antigens / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D015870:Gene Expression; D005796:Genes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010920:Placenta / Q000187:drug effects / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000276:immunology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000941:Antigens; D004274:DNA, Recombinant; D006146:Guanidines; D015849:Plasminogen Inactivators; D019791:Guanidine