Design, Expression, and Purification of a Fusion Enzyme Containing Terminal Deoxynucleotidyl Transferase from B. bovis and DNA-Binding Proteins from E. coli

Background: Gene fusion techniques have yielded promising results in the fusion of thermostable polymerases (Taq and Pfu) with single-stranded and double-stranded DNA-binding proteins. Constructing a terminal deoxynucleotidyl transferase (TdT) fusion enzyme with DNAbinding protein domains can enhance thermostability and broaden the enzyme's application field. This makes it a promising candidate for cost-effective de novo DNA synthesis and a more effective tool for demonstrating apoptosis and detecting viral DNA/RNA. Methods: The design of fusion proteins was based on molecular dynamics and homology modeling. Native and fusion proteins were isolated using affinity chromatography on HisTrap HP. Thermostability was assessed through differential scanning fluorimetry and dynamic light scattering. HPLC analysis was conducted to evaluate enzyme activity. objective: The objectives of this study are to fuse the TdT and EcSSB genes using flexible linkers, to express and purify the resulting fusion proteins, and to investigate their properties and functional activities Results: According to the in silico predictions of the fusion protein structure, a homotetramer was formed. The expressed fusion proteins were successfully purified under native conditions, similar to TdT. The total yields of the studied proteins were 130 mg/L for single-stranded binding protein from E. coli (EcSSB), 5 mg/L for TdT, 9 mg/L for TdT_L1_EcSSB, and 7 mg/L for TdT_L2_EcSSB. The measured radius of TdT (3.5 nm) was found to be consistent with a monomeric structure; however, the fusion proteins were expected to form a homotetramer. Additionally, fusion with EcSSB was found to prevent aggregation, which positively affected the thermal stability of the fusion protein. Instead of elongating the substrate by adding nucleotides, the fusion enzyme removed a nucleotide, specifically TTP, from the 3'-end of the DNA strand. Conclusion: The fusion of TdT with EcSSB resulted in increased thermal stability and a reduced ability to add nucleotides to the substrate.