重组虎纹捕鸟蛛毒素-Ⅰ在巴氏毕赤酵母中的表达及纯化

Expression and Purification of Recombinant Huwentoxin-Ⅰ in Pichia pastoris

虎纹捕鸟蛛毒素Ⅰ是从虎纹捕鸟蛛粗毒中分离纯化 ,具有镇痛活性的肽类神经毒素。对巴氏毕赤酵母生产的重组HWTX Ⅰ进行多步纯化 ,首先将分泌到培养上清的rHWTX Ⅰ进行 90 %饱和度的 (NH4) 2 SO4沉淀 ,再用截留分子量 3kD的滤膜脱盐 ,再用CM阳离子交换层析分离 ,最后用C1 8反相层析脱盐纯化 ,真空干燥后得到的rHWTX Ⅰ经TricineSDS PAGE ,质谱鉴定 ,氨基酸组成分析 ,N 端序列测定及活性鉴定 ,证明已获得高纯度的重组HWTX Ⅰ ,摇瓶表达量约为 80mg L ,约占总分泌量的 2 3.6 % ,并对摇瓶发酵条件进行了优化 ,为利用基因工程方法生产HWTX

HWTX Ⅰ is a peptide neurotoxin purified from the crude venom of the Chinese bird Spider Selenocosmia Huwena, which has analyesic activity. rHWTX Ⅰ expressed by P. pastoris and secreted to culture supernatant was first precipitated by (NH 4) 2SO 4, then it was isolated and desalted by ultrofiltration following by ion exchange chromatography of CM column ,after reverse phase HPLC of C 18 column and vacuum drying, the pure HWTX Ⅰ protein was obtained which was proved to be recombinant HWTX Ⅰ by Tricine SDS PAGE, MALDI TOF mass spectrometry, amino acid composition analysis, the N terminal amino acid sequence and its biological activity. The final yield of the purified HWTX Ⅰ was about 80mg/L accounting for 23 6% of its total secretory proteins.