Proteins are abundant biomolecules that regulate many of the functions developed by cells. Genetically-encoded fluorescent proteins (FPs) have revolutionized our ability to image and study proteins inside live cells and organisms. FPs allow us to monitor the dynamics and to dissect the biological
events associated with specific proteins.
After the discovery of GFP, several FPs with various excitation and emission wavelengths are available and regularly used in bioimaging. However, FPs are relatively bulky (over 200 amino acids, around 30
KDa). This large size of FPs, which are often similar to or even bigger than the protein of interest, can perturb the function, dynamics and localization of labelled proteins. This is particularly critical in small proteins, such as the cytokine IL-33 (18 KDa), a protein with key roles in the immune system, promoting
allergy, inflammation, and resistance to infection. IL-33 is implicated in the development of multiple chronic diseases and metabolic disorders, which disproportionately affect older people and prevent healthy aging across the time course. The biology of IL-33 is not fully understood at the biomolecular level, partially due to the poor specificity of commercially-available reagents. Therefore, there is need for new methods to label small proteins, such as IL-33, in a non-perturbative manner, to enable dynamic and quantitative imaging assays with biomolecular resolution. This technological need drives
the development of this project.
In this project, we will develop the next generation of tools to introduce sitespecifically minimal fluorescent tags (MINITAGS) as small as one single amino acid (0.3 KDa) into proteins using our
recently developed Trp-BODIPY fluorogenic amino acid . In addition to its small size and non-perturbative effect in peptides and proteins, the fluorescence emission of Trp-BODIPY will provide a direct readout of target binding in real time. We will incorporate Trp-BODIPY into IL-33 to
perform bioimaging studies -including superresolution microscopy- with enhanced biomolecular resolution, which are not possible to date.
We will study Trp-BODIPY-IL-33 to assess whether its biological functions are unaffected by the addition of a MINITAG, including affinity of binding to its receptor, cell signalling and induction of immune responses (e.g. SPR, ELISA). We will treat human and murine mast cells and type 2 innate lymphoid cells in vitro with Trp-BODIPY IL-33 (in comparison to wild-type IL-33) to demonstrate responses to IL-33 receptor signalling (e.g. cytokine production measured by ELISA, surface marker expression measured by flow cytometry, and signalling pathways within responsive cells) are maintained. We will also image Trp-BODIPY-IL-33-treated cells to visualize direct cellular uptake and transport of IL-33, including binding specificity assays to IL-33R-positive cells.
MINITAGS have the potential to revolutionise the preparation of small fluorescent proteins for bioimaging at high resolution. In this project, we will develop a revolutionary technology for nonperturbative labelling of proteins to advance basic discoveries in life and health sciences.
 Nat. Commun. 2016, 7, 10940.
 Immunity, 2017, 47, 739.
 Immunol. Cell Biol. 2018, doi: 10.1111/imcb.12200.