虎纹捕鸟蛛毒素XI(HWTX-XI)突变体的真核表达及活性鉴定

Expression and characterization of Huwentoxin-XI(HWTX-XI) and its mutants

虎纹捕鸟蛛毒素-XI(HWTX-XI)是从虎纹捕鸟蛛粗毒中分离的含55个氨基酸残基的蛋白质,兼有胰蛋白酶抑制活性和电压门控钾离子通道抑制活性。通过突变HWTX-XI上的钾离子通道抑制活性关键氨基酸残基设计了2个突变体(分别突变以下氨基酸残基:R5I,R10T,R25A和R5I,R25A),利用pVT102U/α表达载体在酿酒酵母S78中成功表达并获得了高纯度的重组蛋白质;通过分光光度计比色法、膜片钳技术和小鼠脑室注射分别比较三者的胰蛋白酶和钾通道抑制活性以及动物毒性,结果显示:HWTX-XI突变体与HWTX-XI有相同胰蛋白酶抑制活性,但其钾通道活性降低约30倍;小鼠脑室注射HWTX-XI的半致死剂量为247.3μg/kg体重,而突变体在剂量达到25 mg/kg时仍未见明显毒性反应。获得了胰蛋白酶抑制活性保留,但钾通道抑制活性和动物毒性大大降低的突变体,为HWTX-XI胰蛋白酶抑制活性的应用奠定了基础。

Huwentoxin-XI（HWTX-XI） is a protein isolated from the crude venom of spider Ornithoctonus huwena.It has 55 amino acid residues containing 6 cysteine residues forming 3 disulfide bonds.It shows potent inhibitory effect on trypsin and voltage-gated potassium channels in rat dorsal root ganglion cells.According to the structure-function relationship of HWTX-XI,we designed two mutants through mutation of potassium channel inhibition related amino acid residues（R5I,R10T,R25A and R5I,R25A） and then expressed them with high purity by using the vector pVT102U on Saccharamyces cerevisiae strain S78;The two mutants had the same trypsin inhibition activity as HWTX-XI,whereas their potassium channel inhibition activity and animal toxicity were much lower than those of HWTX-XI.This study is helpful for designing drugs of trypsin related diseases based on HWTX-XI.