新型Argonaute蛋白的挖掘及体外活性表征  

The Mining and in Vitro Characterization of Novel Argonaute

作　　者：方蒙君 司振军[1] 沈培杰 黄迪 徐志南[1] FANG Mengjun;SI Zhenjun;SHEN Peijie;HUANG Di;XU Zhinan(College of Chemical&Biological Engineering,Zhejiang University,Hangzhou 310000,China)

机构地区：[1]浙江大学化学工程与生物工程学院,浙江杭州310000

出　　处：《海南师范大学学报（自然科学版）》2023年第4期440-446,共7页Journal of Hainan Normal University（Natural Science）

基　　金：国家重点研发计划项目(2023YFC3040400)。

摘　　要：本研究旨在挖掘新的具有可编码核酸切割活性的Argonaute(Ago)核酸酶。利用已报道的Ago核酸酶的氨基酸序列在NCBI中进行序列比对获得同源性较高的新型Ago蛋白作为候选蛋白;以大肠杆菌为异源表达系统,对候选蛋白的基因进行密码子优化;表达后的目的蛋白利用亲和层析进行分离和纯化;设计核酸定向切割体系以测试纯化蛋白的可编码核酸酶活性。结果显示,序列比对共获得4个候选Ago蛋白,均可在大肠杆菌中可溶表达,再通过亲和层析得到了高纯度的目的蛋白;纯化后的4个候选Ago蛋白经测试均具有可编码核酸内切酶活性。本研究建立的Ago核酸酶的挖掘和体外活性表征的方法,可高效获取新型Ago,对于后继Ago核酸酶的研究工作具有借鉴和参考价值。This study aimed to explore new Argonaute(Ago)nucleases with programmable nucleic acid cleavage activity.The reported Ago nucleases were aligned in NCBI to obtain homologous new Ago proteins.Codon optimization of candidate genes was conducted according to the Escherichia coli heterologous expression system.The expressed target proteins were separated and purified using affinity chromatography.A nucleic acid cleavage system was designed to test the programma⁃ble endonuclease activity of the purified proteins.Results showed that four Ago proteins were chosen by sequence alignment in NCBI,all of which could be expressed solubly in Escherichia coli cells.The expressed Ago proteins were purified using affinity chromatography,resulting in high purity of the target proteins.All four purified Ago proteins were found to possess programmable endonuclease activity.The methods established in this study for the discovery and in vitro characterization of Ago nucleases provide a valuable inspiration for future research on novel Ago nucleases.

关 键 词：Argonaute蛋白 可编码核酸内切酶 核酸酶活性表征 

分 类 号：Q522[生物学—生物化学]