Abstract:[Background] Assembling large DNA fragments is crucial in genome synthesis. Exploring inexpensive and efficient DNA assembly technologies has been a research hotspot in synthetic biology. In some bacteria such as Streptomyces lividans, there exists phosphorothioation modification on DNA (also known as DNA sulfur modification), while in some other bacteria such as Streptomyces coelicolor, there is a protein possessing sulfur-binding domain (SBD), which serves as a reader protein to specifically recognize the sulfur modification on DNA. This inspired us to develop a new technology to ligate long DNA fragments. [Objective] In this study, we introduced sulfur modification at the 5′ and 3′ ends of the DNA fragments to be ligated, and employed a fusion protein containing SBD domain and T4 DNA ligase to complete the ligation of long DNA fragments in vitro and compare the different efficiency between 2.5 kb and 8 kb fragments with these two ligases. [Methods] We designed sulfur-modified primers and amplified 2.5 kb and 8 kb DNA fragments with sulfur modification. Additionally, we constructed three fusion protein vectors: T4-linker-SBD (Hga), T4-linker-SBD (Spr), and T4-linker-SBD (Mmo) and expressed them in Escherichia coli and compared the ligation efficiency between T4 DNA ligase and fusion proteins under three different concentration gradients: 2.4, 0.24, 0.024 mg/mL. [Results] We successfully amplified 2.5 kb and 8 kb DNA fragments with sulfur modification and expressed these three fusion proteins. In the 2.5 kb DNA fragments ligation experiment, we found that T4 DNA ligase and fusion protein under the three concentration gradients and in twelve groups all successfully ligated, whereas in the 8 kb DNA fragments ligation experiment, only T4-linker-SBD (Hga) fusion protein successfully ligated two 8 kb fragments into 16 kb fragment. [Conclusion] According to our agarose gel electrophoresis, we concluded that in the 8 kb DNA fragments ligation experiment, T4-linker-SBD (Hga) fusion protein had higher ligation efficiency. Thus our method provide a new way to assemble large DNA fragments with low cost and high efficiency.