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Functional Expression of a Blue Fluorescent Protein - Photoactive Yellow Protein Fusion in HEK293 and E. coli

Photocontrol, the use of light-sensitive proteins to control events within living tissue, allows complex processes in higher organisms to be studied. The Halorhodospira halophila photoactive yellow protein (PYP) can be used to regulate transcription factor activity with blue light. Before any PYP-based system can probe complex processes in higher organisms, proof of functional expression in vivo is required. We linked d25 PYP to the C-terminus of blue fluorescent protein (BFP) and expressed variants of the fusion protein (BFPd25PYP) in E. coli and human embryonic kidney (HEK293) cells. Expression of BFPd25PYP in E. coli verified in vitro photoswitching. The fusion protein was successfully expressed in HEK293. Fluorescence studies of intact cells indicated chromophore uptake and incorporation into PYP in HEK293, while photoswitching of PYP was measured in protein isolated from HEK293. These findings are promising for the development of applications using PYP for in vivo mammalian photocontrol of biological events.

Identiferoai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/43351
Date11 December 2013
CreatorsYin, Lori Hang
ContributorsWoolley, Andrew
Source SetsLibrary and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada
Languageen_ca
Detected LanguageEnglish
TypeThesis

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