State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Shanghai, China.
Biotechnol J. 2024 Sep;19(9):e2400226. doi: 10.1002/biot.202400226.
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 T 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.
末端脱氧核苷酸转移酶（TdT）是一种独特的 DNA 聚合酶，能够在无模板的情况下将核苷酸掺入单链 DNA 中，这促进了各种基于寡核苷酸的工具和方法的发展，特别是在无模板酶促 DNA 合成领域。然而，在大肠杆菌中表达脊椎动物来源的 TdT 会使纯化变得复杂，并增加生产成本。在本研究中，通过对 TdT 的 N 端截短来提高其表达和稳定性。结果表明，N 端截短可以提高六种 TdT 的表达水平。在截短的突变体中，去除 140 个氨基酸的 N-140-ZaTdT 和 N-140-CpTdT 的蛋白表达水平分别提高了 9.5 倍和 23 倍，明显高于野生型。重要的是，截短保留了 TdT 的催化功能。此外，N-140-ZaTdT 的 T 值增加了 4.9°C。截短突变体的表达改善使其更适合降低生产成本和推进酶工程。
