Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification
| Autor: | John M. Christie, Andrew J. Roe, L. Evan Reddick, Mads Gabrielsen, Neal M. Alto, Jayde A. Gawthorne, Katherine S. H. Beckham, Snezhana Akpunarlieva |
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| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Proteases
Applied Microbiology lcsh:Medicine Gene Expression Golgi Apparatus Biology medicine.disease_cause Kidney Biochemistry Microbiology Green fluorescent protein 03 medical and health sciences Viral Proteins Plasmid medicine Genetics Escherichia coli Animals Cloning Molecular lcsh:Science 030304 developmental biology 0303 health sciences Multidisciplinary Cell fusion Flavoproteins 030306 microbiology Effector Escherichia coli Proteins lcsh:R 3C Viral Proteases Kidney metabolism Proteins Bacteriology Recombinant Proteins Cell biology Rats Luminescent Proteins Cysteine Endopeptidases Cell culture lcsh:Q Gene Function Genetic Engineering Research Article Biotechnology |
| Zdroj: | PLoS ONE PLoS ONE, Vol 7, Iss 12, p e52962 (2012) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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