Heterologous Expression And Electrophysiological Activity Research Of Chilobrachys Jingzhao Spider Toxins

Chilobrachys jingzhao is a venomous tarantula spider living in southern China. At present, more than 60 kinds of toxic peptides have been characterized from its venom, which contains a large variety of compounds. Jingzhaotoxin-III(JZTX-III) is one of the isolated and well studied peptide neurotoxin. JZTX-III, a 36-residue peptide containing three pairs of disulfide bonds, selectively inhibits the h Kv2.1 and h Nav1.5 channels, which are mainly expressed in cardiac myocytes. This gives it considerable research value. JZTX-III is typically isolated from native venom, but its yield is often insufficient to meet research demands. And it has not yet been expressed by prokaryotic expression systems. Here we utilized a galactose auto-induction system to assist the E. coli strain SHuffle T7 Express to express a larger quantity of recombinant JZTX-III(r JZTX-III). After optimizing the induction conditions, we induced the expression of r JZTX-III at 30 ℃, 220 rpm for 16 h. Through Ni-NTA and RP-HPLC purification, the product of r JZTX-III reached 12.1 mg/L. Mass spectrometry result suggested that the molecular weight of the expressed r JZTX-III is correct, confirming that the expression succeeded. Subsequent electrophysiological experiments showed that the r JZTX-III has significant inhibitory effect on h Nav1.5. To further test the applicability of the expression strategy we used here, r JZTX-46 was also expressed by the galactose auto-induction method afterwards. JZTX-46, whose biofunctions remain unclear, is a toxin discovered from the DNA library established on the venom of Chilobrachys jingzhao tarantula. It is made of 30 amino acid residues and contains 3 pairs of disulfide bonds. Through mass spectrometry analysis, the molecular weight of the expressed and purified r JZTX-46 matched its theoretical value. This result suggested that the r JZTX-46 was successfully expressed, and it also suggested that the strategy, which using galactose auto-induction system to help the SHuffle strain express peptide rich in disulfide bonds, had a broad range of applicability. Because JZTX-46 is homologous with JZTX-XII, we measured its electrophysiological activities on h Nav1.5 and h Nav1.7 by referring to the biofunctions of JZTX-XII. But the results were unremarkable. We will test whether JZTX-46 has effects on potassium channels and other types of channels in our future works. In this study, we particularly described the producing process of Ulp protease, which is responsible for the cleavage of the r JZTX-III and r JZTX-46 fusion protein. Due to its high market price, we alternatively obtained pure Ulp protease by prokaryotic expression method. Through enzymatic digestion experiment, 1 μl Ulp protease was capable of digesting 33 μg of substrate at 30 ℃ in 1 h, showing that the produced Ulp protease had a high activity.