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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
Li et al. (Sun,) studied this question.