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Publication Detail
ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant.
  • Publication Type:
    Journal article
  • Publication Sub Type:
  • Authors:
    Halliwell LM, Jathoul AP, Bate JP, Worthy HL, Anderson JC, Jones DD, Murray JAH
  • Publication date:
  • Journal:
    Biotechnology and bioengineering
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  • Print ISSN:
  • Language:
  • Addresses:
    School of Biosciences, University of Cardiff, Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK.
The bright bioluminescence catalysed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bioimaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within 6 loop structures of the bright and thermostable x11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding x11 Fluc activity was largely tolerant to deletions. Targeting an 'omega-loop' motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin, altered emission colours and altered substrate specificity for redshifted analogue DL-infraluciferin. Experimental and in silico analyses suggested remodelling of the XXX-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox. This article is protected by copyright. All rights reserved.
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