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Light activated inteins

Light activated inteins
Light activated inteins
The ability to control posttranslational assembly of proteins would be a powerful research tool that would allow researchers to selectively control and induce the function of a targeted protein. Several approaches have utilized inteins, protein domains that when inserted into a given protein sequence, excise themselves from the host protein, ligating the host protein fragments together to leave the excised intein and a mature protein. This process, known as protein splicing, would be a useful tool for studying protein function if it was selectively inducible. Herein we detail our efforts towards the development of a light activated intein that preferentially undergoes protein splicing in the presence of blue light. Our design combines the LOV2 domain from Avena sativa and the Npu DnaE trans intein from Nostoc punctiforme PCC73102 to yield a light activated intein. It was observed by western blot that this light activated intein demonstrated a 1.74 fold increase in intein mediated protein splicing when exposed to blue light. Mutations to eliminate the protein splicing ability of the light activated intein demonstrated that the extein products observed were the result of intein mediated protein splicing. Mutations to render the LOV2 domain insensitive to light indicated that the LOV2 domain influenced the protein splicing ability of the light activated intein, allowing it to progress at a higher rate in the presence of blue light.
Jones, Dale
91e7e9c4-ee15-4cb6-bafc-6c81b519ecd3
Jones, Dale
91e7e9c4-ee15-4cb6-bafc-6c81b519ecd3
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2

(2015) Light activated inteins. University of Southampton, Chemistry, Doctoral Thesis, 196pp.

Record type: Thesis (Doctoral)

Abstract

The ability to control posttranslational assembly of proteins would be a powerful research tool that would allow researchers to selectively control and induce the function of a targeted protein. Several approaches have utilized inteins, protein domains that when inserted into a given protein sequence, excise themselves from the host protein, ligating the host protein fragments together to leave the excised intein and a mature protein. This process, known as protein splicing, would be a useful tool for studying protein function if it was selectively inducible. Herein we detail our efforts towards the development of a light activated intein that preferentially undergoes protein splicing in the presence of blue light. Our design combines the LOV2 domain from Avena sativa and the Npu DnaE trans intein from Nostoc punctiforme PCC73102 to yield a light activated intein. It was observed by western blot that this light activated intein demonstrated a 1.74 fold increase in intein mediated protein splicing when exposed to blue light. Mutations to eliminate the protein splicing ability of the light activated intein demonstrated that the extein products observed were the result of intein mediated protein splicing. Mutations to render the LOV2 domain insensitive to light indicated that the LOV2 domain influenced the protein splicing ability of the light activated intein, allowing it to progress at a higher rate in the presence of blue light.

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More information

Published date: 31 March 2015
Organisations: University of Southampton, Chemistry

Identifiers

Local EPrints ID: 382905
URI: http://eprints.soton.ac.uk/id/eprint/382905
PURE UUID: 5fba4591-fd9a-4e97-b26e-057adcb1e874
ORCID for Ali Tavassoli: ORCID iD orcid.org/0000-0002-7420-5063

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Date deposited: 05 Nov 2015 14:45
Last modified: 19 Sep 2018 04:01

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