Nature-inspired engineering of an artificial ligase enzyme by domain fusion

Cher Ling Tong, Nisha Kanwar, Dana J Morrone, Burckhard Seelig

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature's concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature's successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.

Original languageEnglish (US)
Pages (from-to)11175-11185
Number of pages11
JournalNucleic acids research
Issue number19
StatePublished - Oct 28 2022

Bibliographical note

Publisher Copyright:
© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.


  • Ligases
  • Protein Engineering/methods
  • Proteins

PubMed: MeSH publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Journal Article
  • Research Support, N.I.H., Extramural


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