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

doi:10.13523/j.cb.20151205

中国生物工程杂志

2015, Vol. 35

Issue (12): 30-36 DOI: 10.13523/j.cb.20151205

研究报告

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

阮景军^1,2, 杨毅¹, 唐自钟¹, 陈惠¹

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

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

RUAN Jing-jun^1,2, YANG Yi¹, TANG Zi-zhong¹, CHEN Hui¹

1. College of Life, Sichuan Agricutural University, Ya'an 625014, China;
2. College of Biological Science and Technology, Wuhan Institute of Biological Engineering, Wuhan 430415, China

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摘要：

目的:为了研究胰蛋白酶抑制剂的活性位点,揭示FtTI结构与功能的关系。将FtTI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1.62nM,1.69 nM,1.9 nM,1.8 nM (BApNA作为底物)。结果:SDS-PAGE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 kDa。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30 min,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FtTI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。

关键词： 苦荞麦; 胰蛋白酶抑制剂; 抑制活性; 定点突变; 表达

Abstract:

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 min, 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.

Key words: Trypsin inhibitor Expression Tartary buckwheat Site-directed mutagenesis Inhibitory activity

收稿日期: 2015-07-20 出版日期: 2015-12-22

ZTFLH:

Q754

基金资助:

四川省科技厅国际合作项目(2010hh0040)资助项目

通讯作者: 陈惠 E-mail: chen62hui@aliyu.com

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引用本文:

阮景军, 杨毅, 唐自钟, 陈惠. 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[J]. 中国生物工程杂志, 2015, 35(12): 30-36.

RUAN Jing-jun, YANG Yi, TANG Zi-zhong, CHEN Hui. Study on Tartary Buckwheat Trypsin Inhibitor Activity Sites by Using Site-directed Mutagenesis. China Biotechnology, 2015, 35(12): 30-36.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20151205 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I12/30

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