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- Title
Engineered DNA ligases with improved activities in vitro.
- Authors
Wilson, Robert H.; Morton, Susan K.; Deiderick, Heather; Gerth, Monica L.; Paul, Hayden A.; Gerber, Ilana; Patel, Ankita; Ellington, Andrew D.; Hunicke-Smith, Scott P.; Patrick, Wayne M.
- Abstract
The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end ‘vector + insert’ cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases.
- Subjects
DNA ligases; BACTERIOPHAGE T4; MOLECULAR biology; CARRIER proteins; BACTERIAL DNA; ESCHERICHIA coli DNA
- Publication
PEDS: Protein Engineering, Design & Selection, 2013, Vol 26, Issue 7, p471
- ISSN
1741-0126
- Publication type
Article
- DOI
10.1093/protein/gzt024