In mammalian cells, the key of ligases for DNA replication was represented to DNA ligase enzyme type 1. It is considered as a vital enzyme and required in recombination and repair processes. Also, the Human DNA ligase enzyme type 1 is essential for encodes as a part of the family of ATP-dependent ligase. According to requirements for ligases function, they are classified into two groups; one group uses ATP such as Eukaryotes and the other needs NAD+ such as bacteria. The main work in the current study is to clone, express and purify Human-DNA Ligase Enzyme type 1 gene (H-Lig1). The H-Lig1 gene consists of an open reading frame containing 2760 bp, which encoded for 919 amino acids residues. This gene was synthesized into an empty plasmid by GeneArt (Life Technologies, Fisher Scientific/UK) and sent as double-stranded DNA with individual plasmid. The key objective of cloning and purification of H-Lig1 is to demonstrate the effect of G-418 Disulfate on Human Lig1 protein activity in vitro, since DNA ligase of prokaryotic cells observe no similarity to the eukaryote ligases. Here in this study it is found that the activity of H-Lig1 protein (represented to Human-DNA Ligase 1) in vitro was not inhibited by using G-418 Disulfate and did not show any inhibition and the half-maximal inhibition concentration of G-418 [IC50] was N/A, which is indicating the prospective DNA ligases as novel antibiotic targets against ligase of bacteria. The results have referred that in spite of DNA ligase in prokaryotes and eukaryotes are functionally similar. However, they are structurally different.