甘蓝型油菜Kunitz蛋白酶抑制剂的异源表达与理化特征分析

利用毕赤酵母表达系统首次克隆表达了一个油菜籽来源的Kunitz蛋白酶抑制剂基因(RTI;rti),分离纯化后对该抑制剂进行了理化特征分析.结果显示:在摇瓶发酵水平,重组RTI诱导表达水平达到628 mg·L-1,抑制剂比活性达到69.6 TIU·mg-1蛋白;重组RTI在30～90℃可保持70％以上抑制活性,在pH 2...

Heterologous expression and physicochemical characterization of Brassica napus Kunitz protease inhibitor

A Kunitz inhibitor gene (RTI;rti)from rapeseed was cloned and expressed in Pichia pastoris expression system for the first time. Its physicochemical characteristics were investigated after separation and purification. The results showed that the expression level of recombinant RTI reached 628 mg·L^-1 and its inhibitory activity reached 69.6 TIU·mg^-1 in shaking flask fermentation. The recombinant RTI maintained over 70% inhibitory activity between 30-90 ℃ and retained over 80% inhibitory activity between pH 2.0-11.0. The metal ions Cu²⁺ and Co²⁺, the organic reagents methanol, ethanol, acetone or chloroform inhibited the activity of RTI. The recombinant RTI reacted with trypsin in a non-competitive manner, and demonstrated a strong and specific inhibitory effect on trypsin. These results indicated that RTI was a typical plant Kunitz trypsin inhibitor. Combined with good physicochemical properties, RTI has the potential to be developed as insect resistant protein.