基于定点突变技术对苦荞麦胰蛋白酶抑制剂活性位点的研究  

Study on Tartary Buckwheat Trypsin Inhibitor Activity Sites by Using Site-directed Mutagenesis

作　　者：阮景军[1,2] 杨毅[1] 唐自钟[1] 陈惠[1] 

机构地区：[1]四川农业大学生命科学学院,雅安625014 [2]武汉生物工程学院生物科学与技术学院,武汉430415

出　　处：《中国生物工程杂志》2015年第12期30-36,共7页China Biotechnology

基　　金：四川省科技厅国际合作项目(2010hh0040)资助项目

摘　　要：目的：为了研究胰蛋白酶抑制剂的活性位点,揭示Ft TI结构与功能的关系。将Ft TI和突变体aFtTI-R65L,aFtTI-D67V和aFtTI-R65L/D67V经IPTG诱导培养5h,收集菌液经过超声波破碎得到粗产物,经过纯化后的胰蛋白酶抑制剂对胰蛋白酶的摩尔抑制比分别为1∶1,1∶1.15,1∶1.3,1∶1.2;抑制常数Ki分别为1.62n M,1.69 n M,1.9 n M,1.8 n M（BAp NA作为底物）。结果：SDSPAGE分析表明突变前和突变后表达产物胰蛋白酶抑制剂的大小一致,均为9.5 k Da。对突变体aFtTI-R65L,aFtTI-D67V和aFtTI-R65L/D67V抑制反应温度研究表明,其最适反应温度均为40℃。在10~80℃保温30 min后,突变体对胰蛋白酶的抑制活性仍保留80%以上;在90℃保温30min,突变体的抑制活性开始显著下降,只保留其39%。具有较高的耐热性。将aFtTI在pH 3.0~10.0的不同缓冲溶液中放置30 min后,其抑制活性可保留90%左右,在pH 2.0条件下,aFtTI抑制活性丧失约31%;在pH 11.0条件下,aFtTI抑制活性丧失约43%。结论：对苦荞麦蛋白酶抑制剂Ft TI的定点突变并不会改变它是一种偏碱性的胰蛋白酶抑制剂的性质,突变前后均保持了耐碱性的特点。Objective： In order to study the active sites of trypsin inhibitor and reveal the relationship between structure and function. Methods：The FtTI gene from tartary buckwheat was mutated by site-directed mutagenesis technology and final three mutant strains R65L, D67V, R65L/D67V were obtained. The expression products were isolated, purified and the inhibition activity measured. The mutants R65L, D67V, R65L/D67V were induced for 5 h by IPTG, inhibition molar ratios are respectively 1： 1, 1： 1.15, 1：1.3 and 1： 1.2. The inhibition constants （Ki） are respectively 1.62nM, 1.69 nM, 1.9 Nm and 1.8 nM （ BApNA as substrate）. Results： The SDS-PAGE analyses of expression products showed that premutation and after mutation of trypsin inhibitor have the same molecular weight as 9.5 kDa. The optimum temperature of mutants＇ aFtTI-R65L, aFtTI- D67V and aFtTI-R65L/D67V is still 40 ℃. The thermal stability results showed that all three mutants have high heat resistance. After 10 - 80 ℃ for 30 min, aFtTI trypsin inhibitory activity remains more than 80%. After 90 ℃ for 30 rain, the aFtTI inhibitory activity begin to decreased significantly and only reserved it＇ s inhibitory activity about 39%. Thus, aFTtI has high heat resistance, aFtTI inhibitory activity could retain about 90% , after placed different buffer solutions （pH 3.0 - 10.0 ） for 30 minutes. Under pH 2.0 conditions, the inhibitory activity of aFtTI loses about 31%. Under pH 11.0 conditions, the aFtTI inhibitory activity loses about 43%. Conclusions： The experiment showed that site-directed mutagenesis of FtTI did not change the alkaline property and maintained the resistance alkali characteristics with or without mutations.

关 键 词：苦荞麦 胰蛋白酶抑制剂 定点突变 表达 抑制活性 

分 类 号：Q754[生物学—分子生物学] Q751