Table 1.
Summary of the proteins used in this study to create the ten iLOV fusions.
Figure 1.
The pET-iLOV plasmid with key features highlighted including the iLOV domain (green) and key restriction sites for cloning genes of interest.
Figure 2.
Heat-map of iLOV fluorescence for the 10 proteins analysed in this study.
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.
Figure 3.
Western blot using an anti-iLOV antibody shows expression of the full-length AdhED2-iLOV-fusion protein (“P3”, A).
Expression of AdhED2-iLOV-fusion protein (“P3”) over time corresponds with fluorescence, (B). AdhED2-iLOV was expressed in E. coli C41 cells and the level of fluorescence monitored. The arrow indicates addition of 1 mM IPTG to induce expression.
Figure 4.
Purification of AdhE-D2-iLOV (P3) is easy to follow due to the distinctive yellow-green colour of the iLOV tagged protein, (A).
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.
Figure 5.
Microinjection of wild-type EspGΔ42 (A), EspG-iLOV (B), and buffer control (C), into cultured NRK cells.
Visualized is the Golgi protein GM130, cascade blue injection dye, and merge as indicated in the vertical columns. Scale bar represents 50 µm.