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 (Ki) 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.