Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech (Hirudo medicinalis)--primary structure, expression in yeast, and characterisation of native and recombinant inhibitor.
We have reported earlier the isolation and amino acid composition of bdellin A from medical leech, and characterised it as an inhibitor of trypsin, plasmin and acrosin [Fritz, H., Gebhardt, M., Meister, R. & Fink, E. (1971) in Proceedings of the international research conference on proteinase inhibitors (Fritz, H. & Tschesche, H., eds) pp. 271-280, Walter de Gruyter, Berlin]. In the present study, one of several chromatographic forms of this inhibitor was isolated from a semi-pure preparation. Elucidation of its amino acid sequence revealed that bdellin A is a member of the antistasin family. Therefore, it was renamed bdellastasin to avoid confusion with bdellin B, which is another trypsin-plasmin inhibitor from the medical leech, but of the Kazal type. Furthermore, a synthetic gene of bdellastasin was constructed, and the protein expressed in Saccharomyces cerevisiae with yields of 29 mg/l. The recombinant bdellastasin was purified by hydrophobic interaction and anion-exchange chromatography. Comparison by mass spectroscopy, far-ultraviolet circular dichroism studies, sequence determination, and inhibition characteristics demonstrated the identity of recombinant and native bdellastasin. The Ki values of bdellastasin for inhibition of bovine trypsin and human plasmin are in the nanomolar range; no inhibition was detected for factor Xa, thrombin, tissue kallikrein, plasma kallikrein and chymotrypsin. Circular dichroism analyses indicated that bdellastasin is devoid of secondary-structural elements.
Moser
M
M
Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität, München, Germany.
Auerswald
E
E
Mentele
R
R
Eckerskorn
C
C
Fritz
H
H
Fink
E
E
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007447:Invertebrate Hormones / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D007865:Leeches / Q000737:chemistry* / Q000235:genetics; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D017931:DNA Primers; D007447:Invertebrate Hormones; D009930:Organic Chemicals; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C086021:hirustasin; C107927:bdellin