摘要
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 2.0~11.0保持80%以上抑制活性;金属离子Cu^(2+)和Co^(2+),有机试剂甲醇、乙醇、丙酮和氯仿对其有抑制作用;重组RTI以非竞争性抑制方式与胰蛋白酶作用,对胰蛋白酶具有很强的、专一性抑制作用,属于典型的植物Kunitz型胰蛋白酶抑制剂。结合其良好的理化特性,重组RTI具有开发为抗虫蛋白的潜力。
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 Cu^(2+) and Co^(2+),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.
作者
冯玮
李朋朋
宋鹏
FENG Wei;LI Pengpeng;SONG Peng(School of Life Sciences, Liaocheng University, Liaocheng 252000, Shandong, China;Bureau of Agricultural and Rural Affairs in Dongchangfu, Liaocheng, Liaocheng 252000, Shandong, China)
出处
《浙江农业学报》
CSCD
北大核心
2022年第1期112-119,共8页
Acta Agriculturae Zhejiangensis
关键词
油菜籽Kunitz蛋白酶抑制剂
毕赤酵母
克隆表达
理化特征
rapeseed Kunitz protease inhibitor
Pichia pastoris
cloning and expression
physicochemical characteristics
作者简介
冯玮(1988—),女,山东聊城人,硕士研究生,主要从事生物活性物质研究。E-mail:fengwei01@lcu.edu.cn;通信作者:宋鹏,E-mail:songpengyg@126.com。
