Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification

Gawthorne, J.A., Reddick, L.E., Akpunarlieva, S.N., Beckham, K.S.H., Christie, J.M. , Alto, N.M., Gabrielsen, M. and Roe, A.J. (2012) Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification. PLoS ONE, 7(12), e52962. (doi: 10.1371/journal.pone.0052962) (PMID:23300834) (PMCID:PMC3531456)

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

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Gawthorne, Ms Jayde and Gabrielsen, Dr Mads and Christie, Professor John and Roe, Professor Andrew
• Authors: Gawthorne, J.A., Reddick, L.E., Akpunarlieva, S.N., Beckham, K.S.H., Christie, J.M., Alto, N.M., Gabrielsen, M., and Roe, A.J.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity; College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: PLoS ONE
• Publisher: Public Library of Science
• ISSN: 1932-6203
• Published Online: 27 December 2012
• Copyright Holders: Copyright © 2012 The Authors
• First Published: First published in PloS ONE 7(12):e52962
• Publisher Policy: Reproduced under a Creative Commons License