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Journal of Biotechnology and Phytochemistry| Volume: 2
October 25-26, 2018 | Frankfurt, Germany
Joint Event
Biotechnology & Medical Microbiology
World Congress on
3
rd
International Conference on
Food Science & Technology
ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single aminoacid
deletion mutations in a thermostable variant light up stem cell therapy for Huntingdon’s disease
in vivo
Amit Paul Jathoul
1
, Mutwakil Abdulla
1
, Lisa M Halliwell
1
, Jack P Bate
1
, Harley L Worthy
1
, D Dafydd Jones
1
, William Gray
1
, James A H
Murray
1
and
James C Anderson
2
1
Cardiff University, United Kingdom
2
University College London, United Kingdom
T
he bright bioluminescence catalysed 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 Flucs:
consecutive single amino acid deletion mutants within six loop
structures of the bright and thermostable × 11Fluc. 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 colours, and altered substrate specificity for
redshifted analog DL-infraluciferin. Experimental and in silico
analyses suggested remodelling 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
paletteof thebiomedical andbiotechnological bioluminescence
enzyme toolbox. Lastly, we constructed a redshifted deletion
mutant which allowed us to track primary stem cells
implanted into rat brain models of Huntingdon’s disease.
Speaker Biography
Amit Paul Jathoul completed his PhD at the age of 29 years from Cambridge University,
United Kingdom. He is a SER CYMRUII Fellow of Cardiff University, United Kingdom. He
has 12 publications including an article having been cited over 160 times, and is currently
inventing/ developing new and exciting tools for bioluminescence imaging.
e:
JathoulA@Cardiff.ac.uk