摘要：Abstract The peptide toxin GsAF II (Kappa-theraphotoxin-Gr2c) is a 31-amino acid peptide, recently isolated from the venom of the tarantula spider Grammostola rosea. The peptide toxin ProTX II (β/ω-theraphotoxin-Tp2a), is a 30-amino acid peptide toxin recently isolated from the venom of the tarantula spider Thrixopelma pruriens. The GsAF II and ProTX II have similar sequence but have an impact on different activities. To find a method of obtaining toxin and to explore whether amino acid sequences affect activities or not, an amino acid mutant was constructed that the first two amino acids are tyr (Y) and cys (C). The YC1 sequence is as follows: YCQKWMWTCDSERKCCEGLVCRLWCKKKIEW. Then, we constructed the YC1 vector (pET-GST-YC1), which was transformed into the Escherichia coli strain SHuffleTM. rYC1 was expressed using auto-induction medium. After using a GST column, the expressed fusion protein was digested using SUMO protease (ULP1) to remove the GST-SUMO tag, and then RP-HPLC and ultrafiltration were used for further purification. rYC1 was further analyzed using SDS-PAGE. Then, the purified rYC1 was verified by MALDI-TOF/TOF mass spectrometry. Finally, the IC50 of rYC1 was determined to be 2.94 μM for the rabbit Nav1.3 (rNav1.3) and the activity is between the ProTX II and GsAF II. Finally, the described method is economical and convenient, and toxins obtained using this method can be used for the study of in channels, neurobiology, pharmacology, or other fields.
关键词：YC1 ; auto-induction ; mass spectrometry ; whole-cell patch-clamp experiments
