An-Na Li, Kun Shi, Bu-Bing Zeng, Jian-He Xu, Hui-Lei Yu
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引用次数: 0
摘要
末端脱氧核苷酸转移酶(TdT)是一种独特的 DNA 聚合酶,可催化核苷酸无模板地掺入单链 DNA,它促进了各种基于寡核苷酸的工具和方法的开发,尤其是在无模板酶法 DNA 合成领域。然而,在大肠杆菌中表达源自脊椎动物的 TdTs 会使纯化复杂化并增加生产成本。本研究对 TdTs 进行了 N 端截短,以提高其表达和稳定性。结果表明,N-末端截短能提高六种 TdTs 的表达水平。在截短的突变体中,N-140-ZaTdT 和 N-140-CpTdT 被去除了 140 个氨基酸,其蛋白表达量比野生型分别高出 9.5 倍和 23 倍。重要的是,截短保留了 TdT 的催化功能。此外,N-140-ZaTdT 的 Tm 值提高了 4.9°C。截短突变体表达能力的提高使它们更适合用于降低生产成本和推进酶工程。
Enhancing the expression of terminal deoxynucleotidyl transferases by N-terminal truncation
Terminal deoxynucleotidyl transferase (TdT), a unique DNA polymerase that catalyzes the template-free incorporation of nucleotides into single-stranded DNA, has facilitated the development of various oligonucleotide-based tools and methods, especially in the field of template-free enzymatic DNA synthesis. However, expressing vertebrate-derived TdTs in Escherichia coli complicates purification and increases production costs. In this study, N-terminal truncation of TdTs was performed to improve their expression and stability. The results revealed that N-terminal truncation could enhance the expression level of six TdTs. Among the truncated mutants, N-140-ZaTdT and N-140-CpTdT, with 140 amino acids removed, exhibited an increase in protein expression, which was 9.5- and 23-fold higher than their wild-types, respectively. Importantly, the truncation preserves the catalytic function of TdT. Additionally, the Tm values of N-140-ZaTdT increased by 4.9°C. The improved expression of the truncated mutants makes them more suitable for reducing production costs and advancing enzyme engineering.
Biotechnology JournalBiochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
8.90
自引率
2.10%
发文量
123
审稿时长
1.5 months
期刊介绍:
Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances.
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