Overexpression and purification of a toxic peptide LaIT2 from Japanese scorpion, Liocheles australasiae

In Japan, there are two species of scorpions, Madara scorpion (Isometrus maculatus) and Yaeyama scorpion (Liocheles australasiae), and both of them are living in Yaeyama island. It has been shown that Liocheles australasiae has venom including β-toxin acting on K⁺-channels (β-KTx) (Juichi et al., 2018) [1]. Interestingly, LaIT2, one of the toxins found in the venom of Liocheles australasiae, displays the virulence for insects but almost not for mammals. Until now, molecular mechanism of the functional specificity of LaIT2 is unknown. To clear this issue, we tried to establish the overexpression system of LaIT2 in Rosetta-gami B (DE3) pLysS, which have trxB/gor mutations to induce the disulfide bond formation. In this study, we have succeeded to overexpress the recombinant LaIT2 (rLaIT2) as a thioredoxin (Trx)-tagged protein, and established the purification protocol with Ni²⁺-NTA column chromatography, enterokinase digestion, and HPLC. We succeeded to obtain approximately 0.5 mg of rLaIT2 from the E. coli cells cultured in 1 L of M9 culture medium. Intramolecular disulfide bonding pattern of rLaIT2 was identified by endopeptidase fragmentation and mass spectrometry. rLaIT2 showed insecticidal activity and antimicrobial activity, and these are almost identical to those of natural LaIT2. ¹H-¹⁵N HSQC spectrum of ¹⁵N-labeled rLaIT2 indicated that the rLaIT2 has a stable conformation.