High-level expression and characterization of two serine protease inhibitors from Trichinella spiralis

• We have expressed two serine protease inhibitors, belonging to the TIL and serpin superfamily.
• Tsp03044 showed inhibitory effects on trypsin, α-chymotrypsin, and pepsin.
• TspAd5 could effectively inhibit the activities of α-chymotrypsin and pepsin.
• Tsp03044 and TspAd5 showed activity between 28 and 48 °C.
• Tsp03044 and TspAd5 were much more highly expressed at the ML phase.

Serine protease inhibitors (SPIs) play important roles in tissue homeostasis, cell survival, development, and host defense. So far, SPIs have been identified from various organisms, such as animals, plants, bacteria, poxviruses, and parasites. In this study, two SPIs (Tsp03044 and TspAd5) were identified from the genome of Trichinella spiralis and expressed in Escherichia coli. Sequence analysis revealed that these two SPIs contained essential structural motifs, which were well conserved within the tumor-infiltrating lymphocytes (TIL) and serpin superfamily. Based on protease inhibition assays, the recombinant Tsp03044 showed inhibitory effects on trypsin, α-chymotrypsin, and pepsin, while the recombinant TspAd5 could effectively inhibit the activities of α-chymotrypsin and pepsin. Both these inhibitors showed activity between 28 and 48 °C. The expression levels of the two SPIs were also determined at different developmental stages of the parasite with real-time PCR. Our results indicate that Tsp03044 and TspAd5 are functional serine protease inhibitors.