Renaturation, purification, and characterization of human plasminogen activator inhibitor type 2 (PAI-2) accumulated at high level in Escherichia coli.
Plasminogen activator inhibitor 2 (PAI-2) is an important regulator of plasminogen activation, which inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). In this study we have developed a high-level expression system by inserting a modified PAI-2 gene downstream of the T7 promoter. The expression level of recombinant PAI-2 amounted to 55-60% of total microbial protein. By efficient renaturation and one-step purification, the recombinant protein was purified to homogeneity. The specific activity and yield of recombinant PAI-2 reached 33,000 IU/mg and 10 mg per gram wet weight of Escherichia coli cells, respectively. The second-order rate constant for uPA was 2.6-2.8 x 10(6) M(-1) x s(-1).
Zhou
A
A
Department of Biochemistry and National Laboratory of Pharmaceutical Biotechnology, Nanjing University, P.R. China.
Jiang
X
X
Dou
F
F
Zhu
D
D
Xu
X
X
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017396:Plasminogen Activator Inhibitor 2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
D017396:Plasminogen Activator Inhibitor 2; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator