Prokaryotic expression of Argonaute protein from Thermus thermophilus and its application value in the detection of KRAS 12D mutation
10.13602/j.cnki.jcls.2025.10.02
- VernacularTitle:嗜热栖热菌Argonaute蛋白的原核表达及其在KRAS 12D突变检测中的应用价值
- Author:
Mingming JIANG
1
;
Fangfang ZHOU
;
Xuemei GU
;
Yanan LI
;
Fengxuan ZHANG
;
Lei WANG
Author Information
1. 上海市第八人民医院检验科,上海 200235
- Publication Type:Journal Article
- Keywords:
Thermus thermophilus;
Argonaute protein;
prokaryotic expression;
protein purification;
nuclease activity;
DNA guide strand
- From:
Chinese Journal of Clinical Laboratory Science
2025;43(10):726-731
- CountryChina
- Language:Chinese
-
Abstract:
Objective To obtain the recombinant Argonaute protein(TtAgo)from Thermus thermophilus using gene cloning and pro-tein purification techniques and detect its endonuclease activity against the KRAS 12D mutation in vitro,thereby to provide experimental evidence for its subsequent application in the detection of gene mutations.Methods The TtAgo gene sequence was amplified from the genome of Thermus thermophilus by the PCR technology.Then,the amplified sequence was inserted into the prokaryotic expression vec-tor pET-28a to construct the recombinant vector pET-28a-TtAgo.After the recombinant TtAgo protein was induced to be expressed,it was purified sequentially by the nickel ion affinity chromatography(Ni-NTA),heparin affinity chromatography,and size-exclusion chromatography.The high-specificity nuclease activity of the recombinant TtAgo protein mediated by a short DNA guide strand(gDNA)was verified in vitro.Results The prokaryotic expression vector pET-28a-TtAgo was successfully constructed by the gene cloning tech-nique,and the soluble expression of the recombinant TtAgo protein was achieved.With the help of protein purification technology,the high-purity recombinant TtAgo protein was obtained.Under the mediation of gDNA,the recombinant TtAgo protein could specifically cleave wild-type single-stranded DNA(ssDNA),double-stranded DNA(dsDNA),and RNA molecules,while had no cleavage activi-ty for tumor KRAS 12D mutant molecules.Conclusion The high-purity recombinant TtAgo protein with nuclease activity is successful-ly obtained,and its targeted cleavage activity towards the KRAS 12D mutation site is detected in vitro.
