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Engineered DNA ligases with improved activities in vitro.

Robert H Wilson¹, Susan K Morton, Heather Deiderick

¹Institute of Natural Sciences, Massey University, Auckland 0632, New Zealand.

Protein Engineering, Design & Selection : PEDS

|June 12, 2013

Summary

Researchers engineered bacteriophage T4 DNA ligase by fusing it with DNA-binding proteins. This enhanced enzyme significantly improved DNA fragment joining, especially for blunt ends, advancing molecular biology techniques.

Keywords:

DNA ligase DNA-binding protein enzyme engineering fusion protein

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Area of Science:

Molecular Biology
Protein Engineering
Biochemistry

Background:

Bacteriophage T4 DNA ligase is crucial in molecular biology for sealing DNA nicks.
Its efficiency is limited in joining double-stranded DNA fragments, particularly blunt ends, impacting experimental success.
Existing limitations necessitate improved DNA ligase activity for advanced molecular biology applications.

Purpose of the Study:

To engineer enhanced DNA ligase activity by fusing T4 DNA ligase with various DNA-binding proteins.
To assess the improved ligase variants' efficacy in joining cohesive- and blunt-ended DNA fragments.
To explore a generalizable protein design strategy for improving DNA ligase function.

Main Methods:

Fusion of T4 DNA ligase with seven distinct DNA-binding proteins (e.g., transcription factors, repair proteins).
Activity screening using agarose gel-based assays for cohesive and blunt-ended fragment joining.
Evaluation of engineered ligases (p50-ligase, ligase-cTF) in blunt-end cloning and library construction for sequencing.

Main Results:

Several fusion proteins significantly enhanced T4 DNA ligase activity, up to 7-fold.
p50-ligase and ligase-cTF demonstrated superior performance in fragment joining assays.
Ligase-cTF improved blunt-end cloning by ~160%; p50-ligase showed similar gains in library construction.
Fusion to p50 also enhanced the activity of Escherichia coli DNA ligase.

Conclusions:

Protein fusion strategy effectively enhances DNA ligase activity for joining DNA fragments.
Engineered ligases like p50-ligase and ligase-cTF offer significant improvements over wild-type enzymes.
The developed protein design approach is generalizable for creating improved DNA ligases for molecular biology.