title: Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification.
abstract: In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an "effector" protein normally produced by enterohemorrhagic E. coli strains and "injected" into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Jayde A
authors.initials: JA
authors.lastname: Gawthorne
authors.forename: L Evan
authors.initials: LE
authors.lastname: Reddick
authors.forename: Snezhana N
authors.initials: SN
authors.lastname: Akpunarlieva
authors.forename: Katherine S H
authors.initials: KS
authors.lastname: Beckham
authors.forename: John M
authors.initials: JM
authors.lastname: Christie
authors.forename: Neal M
authors.initials: NM
authors.lastname: Alto
authors.forename: Mads
authors.initials: M
authors.lastname: Gabrielsen
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Roe
mesh_terms: D000086042:3C Viral Proteases; D000818:Animals; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005420:Flavoproteins / Q000235:genetics* / Q000378:metabolism; D006056:Golgi Apparatus / Q000235:genetics* / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051381:Rats; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C429282:EspG protein, E coli; D005420:Flavoproteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases