Improvement of duplex-specific nuclease salt tolerance by fusing DNA-binding domain of DNase from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix.

IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Wenhao Hu, Jin Wang, Juan Li, Mengxia Yang, Zhixing Li, Xuning Zhang, Fang Wu, Yaqi Zhang, Zhidan Luo, Henghao Xu
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引用次数: 0

Abstract

Salt tolerance is an important property of duplex-specific nuclease (DSN). DSN with high salt tolerance can be more widely used in genetic engineering, especially in the production of nucleic acid drugs. To improve the salt tolerance of DSN, we selected five DNA-binding domains from extremophilic organisms, which have been shown the ability to improve salt tolerance of DNA polymerases and nucleases. The experimental results demonstrated that the fusion protein TK-DSN produced by fusing a N-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs domain from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix, has a significantly improved salt tolerance. TK-DSN can tolerate the concentration of NaCl up to 800 mM; in addition, the ability of digesting DNA was also enhanced during in vitro transcription and RNA purification. This strategy provides the method for the personalized customization of biological tool enzymes for different applications.

Abstract Image

通过融合极耐盐细菌thioalalivibrio sp. K90mix dna结合域提高双特异性核酸酶的耐盐性。
耐盐性是双特异性核酸酶(DSN)的重要特性。具有高耐盐性的DSN可以更广泛地应用于基因工程,特别是核酸药物的生产。为了提高DSN的耐盐性,我们从嗜极生物中选择了5个DNA结合结构域,这些结构域已被证明能够提高DNA聚合酶和核酸酶的耐盐性。实验结果表明,通过融合来自极耐盐细菌thioalalivibrio sp. K90mix的包含两个HhH(螺旋-发夹-螺旋)基序结构域的n端dna结合域产生的融合蛋白TK-DSN显著提高了耐盐性。TK-DSN耐NaCl浓度达800 mM;此外,在体外转录和RNA纯化过程中,消化DNA的能力也得到了增强。该策略为不同用途的生物工具酶的个性化定制提供了方法。
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来源期刊
Extremophiles
Extremophiles 生物-生化与分子生物学
CiteScore
6.80
自引率
6.90%
发文量
28
审稿时长
2 months
期刊介绍: Extremophiles features original research articles, reviews, and method papers on the biology, molecular biology, structure, function, and applications of microbial life at high or low temperature, pressure, acidity, alkalinity, salinity, or desiccation; or in the presence of organic solvents, heavy metals, normally toxic substances, or radiation.
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