Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae)

Characterization of Kunitz-type protease inhibitor purified from hemolymph of Galleria mellonella larvae

“…6) nor trypsinlike protease PR2 isolated from the entomopathogenic fungus, Metarhizium anisopliae (data not shown). Thus far, several Kunitztype SPIs have been detected in a variety of insects, including Manduca sexta (Ramesh et al, 1988), Sarcophaga bullata (Papayannopoulos and Biemann, 1992), Bombyx mori (Nirmala et al, 2001a,b), Drosophila virilis (Kress et al, 2004), Haematobia irritans irritans (Azzolini et al, 2004), and G. mellonella (Nirmala et al, 2001a,b), and the ixodid tick, Haemaphysalis longicornis (Miyoshi et al, 2010). With regard to the physiological functions of insect Kunitz SPIs, it has been proposed that a Kunitz SPI from the midgut of the tick performed a role as an anticoagulant factor upon blood feeding (Corral-Rodríguez et al, 2009), and two inhibitors from the cocoons of B. mori (Kurioka et al, 1999) and G. mellonella (Nirmala et al, 2001a,b) were responsible for the protection of the silk against proteolytic attack by bacterial and fungal proteases.…”

“…Thus far, a number of SPIs have been detected in the hemolymph, the midgut and the silk glands of a variety of insects (Polanowski and Wilusz, 1996;Azzolini et al, 2004;Kurioka et al, 1999). On the basis of the amino acid sequence and the tertiary structure, insect SPIs are classified into several families: Kunitz-type (Laskowski, 1986), Kazal-type (Friedrich et al, 1993), Serpin-type (Bode and Huber, 1992) and pacifastin-related (Breugelmans et al, 2009) SPIs.…”

Validation of a Phage Display Method for Protease Inhibitor Selection Using SFTI and HiTI Synthetic Hybrid Peptides

“…HiTI gene was cloned into the phagemid vector pCANTAB 5E and a random library was constructed with mutations restricted to the positions P1' -P3' and P5' of HiTI reactive site [8,9] as described elsewhere [29]. The HiTI gene was amplified by PCR using the construction pHiTI 9.1 [9] and the oligonucleotides PCTBHIFW, 5'-GGGTATCGGCCGAGAAGGCCTTTGATAAAGCTGAC TGCAGTTTGCCCAAAGAGGTTGGGCCCTGTCGC-3', and PCTBHIRV, 5'-GCGAATTAATTCGCGGCGGCCGC CATGCATGACTGC-3'.…”

“…Recently, HiTI (H. irritans trypsin inhibitor) was isolated from the thorax extract of this fly and characterized as a serine protease inhibitor. Recombinant HiTI, produced in Pichia pastoris, can inhibit endogenous trypsin-like enzymes, and it can also interfere in the activity of Escherichia coli protease Omp-T [8,9], suggesting a role for it in the innate immune response of H. irritans.…”

“…Thus, efforts to increase the knowledge on insect biochemistry have been made to help in the development of new methods to control plagues, including the production of vaccines that contain recombinant antigens [5]. In the last decade, several new proteins have been described in H. irritans, such as anti-hemostatic molecules [6], serine proteases [1,7] and serine protease inhibitors [8,9]. Recently, HiTI (H. irritans trypsin inhibitor) was isolated from the thorax extract of this fly and characterized as a serine protease inhibitor.…”