Cloning, purification and biochemical characterization of dipetarudin, a new chimeric thrombin inhibitor.
The development of thrombin inhibitors could provide invaluable progress for antithrombotic therapy. In this paper, we report the cloning, purification and biochemical characterization of dipetarudin, a chimeric thrombin inhibitor composed of the N-terminal head structure of dipetalogastin II, the strongest inhibitor from the assassin bug Dipetalogaster maximus, and the exosite 1 blocking segment of hirudin, connected through a five glycine linker. The cloning of dipetarudin was performed by a simple method which had not been used previously to clone chimeras. Biochemical characterization of dipetarudin revealed that it is a slow, tight-binding inhibitor with a molecular mass (M(r)=7560) and a thrombin inhibitory activity (K(i)=446 fM) comparable to r-hirudin.
López
M
M
Research Unit Pharmacological Haemostaseology, Medical Faculty at the Friedrich-Schiller-University Jena, Drackendorfer Str. 1, 07747, Jena, Germany.
Mende
K
K
Steinmetzer
T
T
Nowak
G
G
D000991:Antithrombins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D019476:Insect Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity
D000991:Antithrombins; D017931:DNA Primers; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; C407584:dipetalogastin protein, Dipetalogaster maximus; D012697:Serine Endopeptidases