Design and Characterization of the Fusion Enzyme of Bovine Terminal Deoxynucleotidyl Transferase and DNA Binding Protein Sso7d from Sulfolobus solfataricus

The terminal deoxynucleotidyl transferase is a unique polymerase that incorporates nucleotides at the 3'-terminus of single-stranded DNA primers in a template-independent manner. This biological function propels the development of numerous biomedical and bioengineering applications. However, the extensive use of TdT is constrained by its low expression levels in E. coli and low optimal operating temperature. To address these limitations, a fusion enzyme combining TdT and the DNA-binding protein Sso7d from Sulfolobus solfataricus is designed and isolated. This fusion protein exhibits a threefold increase in DNA-binding affinity to double-stranded DNA and demonstrates improved thermostability, denaturing at temperatures up to 48 °C, along with an enhancement in the expression yield of the target protein. The fusion of Sso7d to the C-terminus of TdT enhances catalytic activity for nucleotide removal from the coding end, while completely abolishing nucleotide addition activity. Conversely, the presence of Sso7d at the N-terminus of TdT decreases nucleotide incorporation and enhances nucleotide removal activity, with this effect significantly influenced by substrate length and the presence of metal ions in the reaction mixture. Overall, the obtained fusion enzymes could serve as promising candidates for novel specific applications, such as gene editing, mutagenesis studies, and aptamer engineering.