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

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

Heat-map of iLOV fluorescence for the 10 proteins analysed in this study.

<p>Each protein-iLOV fusion was cultured in four different media (LB, TB, 2YT and M9), three different cell lines (C41, pLysS, Rosetta) and at two different temperatures (25°C and 37°C). Peak fluorescence was determined during the growth curve. Fluorescence data were normalized from the absolute fluorescence values to a scale of 0 to 1. The area of interest was highlighted, and a “surface contour” representation was selected.</p

Microinjection of wild-type EspGΔ42 (A), EspG-iLOV (B), and buffer control (C), into cultured NRK cells.

<p>Visualized is the Golgi protein GM130, cascade blue injection dye, and merge as indicated in the vertical columns. Scale bar represents 50 µm.</p

Western blot using an anti-iLOV antibody shows expression of the full-length AdhED2-iLOV-fusion protein (“P3”, A).

<p>Expression of AdhED2-iLOV-fusion protein (“P3”) over time corresponds with fluorescence, (B). AdhED2-iLOV was expressed in <i>E. coli</i> C41 cells and the level of fluorescence monitored. The arrow indicates addition of 1 mM IPTG to induce expression.</p

Purification of AdhE-D2-iLOV (P3) is easy to follow due to the distinctive yellow-green colour of the iLOV tagged protein, (A).

<p>Purified AdhE-D2 lacking the iLOV domain is colorless, (B). Purified P3 shows fluorescence under ultraviolet light, (C). Purified AdhE-D2 lacking the iLOV domain viewed under uv light (D). The iLOV domain can be readily cleaved from purified proteins using C3 protease, (E). Lane M: Markers, Lane 1: AdhE-D2-iLOV, indicated by arrow, Lane 2: AdhE-D2, iLOV and 3C protease indicated by arrows. The iLOV-His₈ tag has been cleaved off, and the size of AdhE-D2 has been reduced by approximately 16 kDa.</p

The pET-iLOV plasmid with key features highlighted including the iLOV domain (green) and key restriction sites for cloning genes of interest.

<p>The pET-iLOV plasmid with key features highlighted including the iLOV domain (green) and key restriction sites for cloning genes of interest.</p