Enhancing the expression of terminal deoxynucleotidyl transferases by N‐terminal truncation
A Li, Kun Shi, Bu‐Bing Zeng, Jian‐He Xu, Hui‐Lei Yu
Abstract
Abstract 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‐ Za TdT and N‐140‐ Cp TdT, 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 m values of N‐140‐ Za TdT increased by 4.9°C. The improved expression of the truncated mutants makes them more suitable for reducing production costs and advancing enzyme engineering.