Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae.
Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.
Steven
J
J
Delta Biotechnology Ltd, Nottingham, England.
Cottingham
I R
IR
Berry
S J
SJ
Chinery
S A
SA
Goodey
A R
AR
Courtney
M
M
Ballance
D J
DJ
D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors; D015849:Plasminogen Inactivators / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D004247:DNA; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator