Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification

Jayde A. Gawthorne, L. Evan Reddick, Snezhana N. Akpunarlieva, Katherine S H Beckham, John M. Christie, Neal M. Alto, Mads Gabrielsen, Andrew J. Roe

Research output: Contribution to journalArticle

18 Scopus citations

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.

Original languageEnglish (US)
Article numbere52962
JournalPloS one
Volume7
Issue number12
DOIs
StatePublished - Dec 27 2012

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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    Gawthorne, J. A., Reddick, L. E., Akpunarlieva, S. N., Beckham, K. S. H., Christie, J. M., Alto, N. M., Gabrielsen, M., & Roe, A. J. (2012). Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification. PloS one, 7(12), [e52962]. https://doi.org/10.1371/journal.pone.0052962