, 1990); and the thrombin inhibitors ornithodorin (from O. moubata ( van de et al., 1996)), savignin (from Ornithodoros savignyi ( Mans et al., 2002b)), monobin (from Argas monolakensis ( Mans and Ribeiro, 2008)) and boophilin (from R. microplus ( Macedo-Ribeiro et al., 2008)). Boophilin is a doubled-headed Kunitz inhibitor displaying 3-Methyladenine price a P1 Lys residue at the canonical reactive loop of its N-terminal Kunitz domain. However, boophilin inhibits thrombin in a non-canonical manner, inserting its N-terminal segment into thrombin’s active

site, while its C-terminal Kunitz domain binds to the exosite I of the protease ( Macedo-Ribeiro et al., 2008). Given the important role of Kunitz-type inhibitors in the R. microplus life cycle and the high specificity of boophilin for thrombin, we expressed and purified full-length mature boophilin and its N-terminal Kunitz domain in large scale using a Pichia pastoris system. We also profiled boophilin gene expression and evaluated the effect

of RNAi gene silencing in tick egg production. R. (Boophilus) microplus (Babesia spp.-free) ticks were supplied by Dr. Itabajara da Silva Vaz Junior (Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, RS, Brazil). The pPICZαB vector and P. pastoris strain KM71H were purchased from Invitrogen (Carlsbad, CA, USA) and used following the supplier’s instructions. DAPT price DNA sequencing was performed using

the BigDye Terminator V3.1 Cycle Sequencing Kit on an ABI 377 found or ABI 3130 sequencer (Applied Biosystems, Foster City, CA, USA). The substrates S2484 (Pyro-Glu-Pro-Val-pNa) and S2238 (HD-Phe-Pip-Arg-pNa), were purchased from Chromogenix (Molndal, Sweden) and tosyl-Gly-Pro-Arg-pNA from Sigma (Darmstadt, Germany). Bovine trypsin (EC 3.4.21.4) and bovine thrombin (EC 3.4.21.5) were obtained from Sigma (St. Louis, MO, USA) and human neutrophil elastase (EC 3.4.21.37) from Calbiochem (San Diego, CA, USA). RNA from ovary, fat body, salivary gland, gut, and hemocytes of engorged R. microplus adult females was extracted using Tryzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The cDNAs were synthesized using the ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA). Quantitative PCR was performed using two specific primers designed based on the boophilin sequence with GenBank accession number AJ304446: Boophilinfw (5′-CAG AGA AAT GGA TTC TGC CGA CTG CCG GCA-3′) and Boophilinrev (5′-ACA CTC CTC TAT GGT CTC GAA-3′). R. microplus elongation factor 1-alpha (ELF1a) specific primers – ELF1afw (5′-CGT CTA CAA GAT TGG TGG CAT T-3′) and ELF1arv (5′-CTC AGT GGT CAG GTT GGC AG-3′) – were used for DNA amplification control.