Halliwell, LM;
Jathoul, AP;
Bate, JP;
Worthy, HL;
Anderson, JC;
Jones, DD;
Murray, JAH;
(2018)
Delta Flucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant.
Biotechnology and Bioengineering
, 115
(1)
pp. 50-59.
10.1002/bit.26451.
Preview |
Text
Anderson_Biotech Bioeng Accepted version_230817.pdf - Accepted Version Download (395kB) | Preview |
Abstract
The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging 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 six loop structures of the bright and thermostable ×11 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 ×11 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 colors, and altered substrate specificity for redshifted analog DL‐infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω‐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.
Type: | Article |
---|---|
Title: | Delta Flucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant |
Open access status: | An open access version is available from UCL Discovery |
DOI: | 10.1002/bit.26451 |
Publisher version: | http://dx.doi.org/10.1002/bit.26451 |
Language: | English |
Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
Keywords: | bioimaging, bioluminescence, bioreporter, deletion mutagenesis, firefly luci ferase, infraluciferi |
UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
URI: | https://discovery.ucl.ac.uk/id/eprint/10049031 |
Archive Staff Only
View Item |