Ligase, an enzyme that uses ATP to form bonds, is used in recombinant DNA cloning to join restriction endonuclease fragments that have annealed. The ligase commonly used is T4 DNA ligase, which was first isolated from E. coli that were infected with the lytic bacteriophage T4

Ligase Enzyme

Ligase enzyme (also known as DNA ligase) is an essential enzyme involved in the process of DNA replication and repair. It catalyzes the formation of phosphodiester bonds between adjacent DNA fragments, leading to the formation of a continuous DNA strand. The isolation of ligase enzyme has contributed significantly to various molecular biology techniques, such as DNA cloning, PCR, and genetic engineering.

Isolation of Ligase Enzyme

The isolation of ligase enzyme was initially performed from a specific bacterium called Escherichia coli (E. coli). E. coli is a gram-negative bacterium commonly found in the intestines of humans and animals. It possesses a variety of enzymes involved in DNA replication and repair, including ligase enzyme.

The isolation process involves several steps:

1. Cell Culturing: E. coli cells are cultured in a suitable growth medium, which provides essential nutrients for their growth and replication. The cells are allowed to grow under optimal conditions, such as temperature, pH, and oxygen availability.

2. Cell Harvesting: Once the cells have reached the desired growth phase, they are harvested from the culture medium. This can be done by centrifugation, where the cells are separated from the culture medium based on their density.

3. Cell Lysis: The harvested cells are then subjected to cell lysis, where the cell membrane is disrupted to release the intracellular components. This can be achieved by physical methods, such as sonication or freeze-thaw cycles, or by using chemical agents that break down the cell membrane.

4. Protein Extraction: After cell lysis, the protein mixture is obtained, which contains various cellular proteins, including ligase enzyme. To separate the desired enzyme from other proteins, techniques such as ammonium sulfate precipitation, chromatography, or affinity purification can be employed.

5. Enzyme Assay: Once the ligase enzyme is isolated, its activity needs to be assessed. An enzyme assay is performed to determine the specific activity of the ligase enzyme, which indicates how efficiently it catalyzes the ligation reaction.

6. Enzyme Purification: If required, further purification steps can be performed to obtain a highly pure form of the ligase enzyme. This can involve additional chromatography techniques or electrophoresis to remove any remaining impurities.

7. Enzyme Storage: Finally, the isolated and purified ligase enzyme can be stored under appropriate conditions to maintain its stability and activity for future use in various molecular biology experiments.

Overall, the isolation of ligase enzyme from E. coli has been crucial in understanding its structure, function, and application in various molecular biology techniques.