Hybrid beetle luciferases derived from firefly luciferases and murine medium-chain fatty acyl-CoA synthetases for in vivo imaging

Long, James Alexander 2021. Hybrid beetle luciferases derived from firefly luciferases and murine medium-chain fatty acyl-CoA synthetases for in vivo imaging. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The firefly luciferase (Fluc) bioluminescence system has been widely studied and used by biomedical researchers for in vitro assays as well as a wide variety of optical imaging applications in small animals. However, Fluc is immunogenic to mice which limits its applications, especially in longitudinal experiments requiring time courses extending over more than four weeks. Murine acyl-CoA synthetases (ACSs) are structurally and functionally similar to Flucs and as native mouse proteins are inherently non-immunogenic. Both ACS and Fluc enzymes are composed of N and C-terminal domains (NTD and CTD). A strategy of hybridisation between Photinus pyralis (Ppy) Fluc and several medium-chain ACSs (ACSMs) was employed with the aim of creating hybrids with minimal immunogenicity whilst maintaining brightness when ultimately expressed in BALB/c mice. Several hybrid variants were created with regions of importance substituted into an ACSM backbone, but all the constructs tested failed to produce any bioluminescence when expressed in E. coli. Hybrids created containing the NTD of Ppy Fluc and the CTD of murine ACSMs 1, 2, and 3 were named PpA1, PpA2, and PpA3 respectively. Light emission of these hybrids was respectively ∼0.9%, ∼0.004%, and ∼0.2% that of WT Ppy Fluc when expressed in E. coli, but PpA1 was ∼10% and PpA3 was ∼0.2% as bright as when expressed in HEK cells. The PpA1 hybrid was selected as the best candidate and was expressed in immunocompromised NSG™ mice, producing ∼45% the light output of WT Ppy Fluc. Further modificationsto PpA1 included a construct which replaced a major epitope in the Ppy Fluc NTD (G160 – V168) of PpA1 with the equivalent sequence from a highly similar luciferase with lower predicted immunogenicity; when expressed in HEK cells this construct was marginally brighter than PpA1, emitting light with λmax 10 nm red-shifted.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	26 May 2021
Last Modified:	26 May 2022 01:30
URI:	https://orca.cardiff.ac.uk/id/eprint/141585

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