Cloning, purification, crystallization and preliminary X-ray diffraction analysis of the antistasin-type inhibitor ghilanten (domain I) from Haementeria ghilianii in complex with porcine beta-trypsin.
Ghilanten, isolated from the leech Haementeria ghilianii, is a potent two-domain anticoagulant protein homologous to the factor Xa inhibitor antistasin. A synthetic gene encoding the amino-terminal domain of ghilanten (ghilanten-D1) was constructed, expressed in the methylotrophic yeast Pichia pastoris and purified by heparin-Sepharose chromatography. Recombinant ghilanten-D1 inhibits bovine trypsin and human factor Xa with equilibrium inhibition constants (K(i)) of 126 and 1.2 nM, respectively. Ghilanten-D1 has been crystallized in complex with porcine beta-trypsin; three different-looking but isomorphous crystal forms were obtained, each belonging to the orthorhombic space group P2(1)2(1)2(1). These crystals diffracted to beyond 3.6 A resolution using a rotating-anode X-ray source. A data set complete to 3.7 A resolution was collected.
Rester
U
U
Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, 82152 Martinsried, Germany. ulrich.rester@callistogen.com
Bode
W
W
Sampaio
C A
CA
Auerswald
E A
EA
Lopes
A P
AP
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D007447:Invertebrate Hormones / Q000737:chemistry*; D007865:Leeches / Q000737:chemistry*; D008969:Molecular Sequence Data; D012471:Salivary Proteins and Peptides / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014357:Trypsin / Q000737:chemistry*
D007447:Invertebrate Hormones; D012471:Salivary Proteins and Peptides; C063005:ghilanten; D014357:Trypsin