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Updated: May 20, 2025

Production of Double-stranded DNA Ministrings

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Published on: February 29, 2016

DNA Ligase From Thermococcus radiotolerans: Recombinant Production, Characterization, Up-Scale Fermentation, and In

Ahmed Osman¹, Saima Iftikhar², Bibi Nazia Murtaza³

¹Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.

Biotechnology and Applied Biochemistry

|March 26, 2025

Summary

This study details the cloning, expression, and characterization of a novel DNA ligase from Thermococcus radiotolerans. This enzyme shows high activity and stability, making it a promising tool for DNA manipulation.

Keywords:

DNA ligase Thermococcus radiotolerans activity in silico recombinant up‐scale fermentation>

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Last Updated: May 20, 2025

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Published on: November 30, 2018

Area of Science:

Molecular Biology
Enzymology
Biotechnology

Background:

DNA ligases are essential enzymes for DNA replication, repair, and recombination.
Thermophilic enzymes offer advantages in stability and activity for molecular biology applications.

Purpose of the Study:

To clone, express, purify, and characterize a DNA ligase from the thermophilic bacterium Thermococcus radiotolerans (TR).
To evaluate the enzyme's activity, stability, and potential applications in DNA manipulation.

Main Methods:

Molecular cloning and heterologous expression in Escherichia coli.
Protein purification and characterization using SDS-PAGE and enzyme activity assays.
Optimization of culture conditions (pH, temperature, media, inducer) for enzyme production.
In silico analysis of protein structure and function.

Main Results:

Recombinant DNA ligase-TR (44 kDa) was successfully expressed and purified.
The enzyme exhibits optimal activity at 50°C, pH 7, with ATP and NAD+.
DNA ligase-TR demonstrated higher activity than T4 DNA ligase and unique structural features.
In silico studies identified key residues involved in AMP and DNA binding.

Conclusions:

DNA ligase-TR is a novel, highly active, and thermostable enzyme.
Its unique properties suggest significant potential for various DNA manipulation procedures.
This enzyme represents a valuable addition to the toolkit for molecular biologists.