Cloning, purification, crystallization and preliminary X-ray diffraction analysis of the antistasin-type inhibitor ghilanten (domain I) from Haementeria ghilianii in complex with porcine β-­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 β-trypsin; three different-looking but isomorphous crystal forms were obtained, each belonging to the orthorhombic space group P2₁2₁2₁. These crystals diffracted to beyond 3.6 Å resolution using a rotating-anode X-ray source. A data set complete to 3.7 Å resolution was collected.