All these amino acid exchanges occur on the solvent-exposed face of the inhibitor on its complex with thrombin ( Macedo-Ribeiro et al., 2008) and are therefore unlikely

to affect its learn more anticoagulant activity. Full-length boophilin and D1 were expressed in P. pastoris at high levels (21 and 37.5 mg/L, respectively) and purified by affinity chromatography on trypsin-Sepharose ( Fig. 2A and B). On SDS-PAGE, purified boophilin displayed an apparent molecular mass of 20 kDa and purified D1 of 11 kDa ( Fig. 2C). The inhibitory activity of boophilin against thrombin, trypsin and neutrophil elastase was assessed, and the corresponding inhibition constants (Ki) determined ( Table 1). Purified boophilin showed high selectivity to thrombin with a Ki of 57 pM, a value significantly lower than the 1.80 nM reported for boophilin produced in Escherichia coli ( Macedo-Ribeiro et al., 2008). The second Kunitz domain of boophilin displays an alanine residue at the reactive loop P1 position ( Schechter and Berger, 1967), suggesting it could inhibit elastase.

Both boophilin and D1 inhibited human neutrophil elastase in vitro with Ki values of 21 nM and 129 nM, respectively. Boophilin inhibits thrombin by binding simultaneously to the active site and the exosite 1 of the protease ( Macedo-Ribeiro et al., 2008). The contribution of the interaction with the exosite 1 to the inhibitory activity of boophilin was probed by comparing its activity towards α-thrombin and the exosite 1-less form, γ-thrombin next ( PLK inhibitor Fig. 3). Recombinant boophilin revealed no activity towards γ-thrombin, in amounts that completely abolished

the amydolytic activity of α-thrombin, therefore underscoring the importance of the interaction with the exosite 1. Different tissues of engorged R. microplus females were dissected and used for total RNA purification and cDNA synthesis ( Fig. 4). Boophilin gene expression was mostly detected in the midgut (25,000 fold above other tissues) with minor expression levels in hemocytes, although a contamination with midgut cells during dissection cannot be discarded. In an attempt to unveil boophilin’s physiological role, a RNAi-mediated gene silencing experiment was performed. Three groups of ticks, each composed of 25 animals, were injected with either boophilin dsRNA, PBS buffer or left untreated. In comparison to the control animals, an efficient silencing of boophilin expression was achieved after boophilin dsRNA treatment (Fig. 5A). Boophilin down-regulation resulted in a decrease (∼20% after 24 and 48 h) in egg production (Fig. 5B). Considering the important role of Kunitz-type inhibitors in the life cycle of R. microplus and the high specificity of the tandem Kunitz inhibitor boophilin for thrombin, full-length boophilin and its N-terminal Kunitz domain (D1) were expressed, purified and characterized.