Ruan Jingjun, Yan Jun, Hou Shengqi, Chen Hui, Wu Qi, Han Xueyi
College of Life Sciences, Sichuan Agriculture University, Ya'an, 625014, Sichuan, People's Republic of China,
Mol Biol Rep. 2015 Jan;42(1):209-16. doi: 10.1007/s11033-014-3760-y. Epub 2014 Sep 26.
The gene of the trypsin inhibitor of tartary buckwheat (Fagopyrum tataricum) was successfully cloned, expressed in Pichia pastoris and tested for regulatory effects on insect growth. The three significant factors were optimized by single-factor experiments and central composite design in response surface methodology. Proteins were efficiently expressed at levels of 489.6-527.4 U/mg in shaken flasks. The trypsin inhibitor from tartary buckwheat (FtTI) was purified by affinity chromatography and centrifugal ultrafiltration. The purified FtTI efficiently inhibited trypsin protease activity by competitive inhibition with a Ki value 1.5 nM. The molecular mass of the purified protein was approximately 13.8 kDa. FtTI had a higher toxic killing effect on Mamestra brassicae larvae. The median lethal concentration for the larvae was 15 μg/mL.
苦荞（鞑靼荞麦）胰蛋白酶抑制剂基因被成功克隆，并在毕赤酵母中表达，同时对其对昆虫生长的调控作用进行了测试。通过单因素实验和响应面法中的中心复合设计对三个显著因素进行了优化。在摇瓶中蛋白质的有效表达水平为489.6 - 527.4 U/mg。采用亲和色谱和离心超滤法对苦荞胰蛋白酶抑制剂（FtTI）进行纯化。纯化后的FtTI通过竞争性抑制有效抑制胰蛋白酶的蛋白酶活性，其Ki值为1.5 nM。纯化蛋白的分子量约为13.8 kDa。FtTI对甘蓝夜蛾幼虫具有较高的毒杀作用。幼虫的半数致死浓度为15 μg/mL。
