Expression, isolation and characterization of recombinant [Arg15,Glu52]aprotinin.
The [Arg15,Glu52]aprotinin gene has been constructed from a synthetic [Glu52]-aprotinin gene via an exchange of the appropriate DNA cassette. The gene has been fused to the N-terminal part of the bacteriophage MS-2 polymerase and expressed in a temperature inducible E. coli expression system. The produced fusion protein is deposited as inclusion bodies. Pure and functionally active [Arg15,Glu52]aprotinin has been obtained after cleavage of the purified fusion protein and renaturation of the aprotinin homologue. Recombinant [Arg15,Glu52]aprotinin shows good inhibition of human anionic and cationic trypsin (Ki less than or equal to 10(-11)M) and of human plasma kallikrein (Ki = 3.2 x 10(-10)M). The inhibition constants for human plasmin are Ki = 1.3 x 10(-10)M and for human urinary kallikrein Ki = 10(-11)M. No inhibition was found with the human proteinases thrombin, coagulation factor Xa, urokinase, tissue plasminogen activator, cathepsin G, leukocyte elastase and pancreatic elastase.
Auerswald
E A
EA
Bayer AG, Institut für Biochemie, Wuppertal.
Hörlein
D
D
Reinhardt
G
G
Schröder
W
W
Schnabel
E
E
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000032:analysis* / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors; D012967:Sodium Dodecyl Sulfate
D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C057845:aprotinin, Arg(15)-Glu(52)-; D012967:Sodium Dodecyl Sulfate; D007611:Aprotinin