Bioluminescent enzymes are widely used for biomedical imaging as genetic reporters.
Unlike other techniques, bioluminescence imaging allows for “seeing” cells and diseases in
living organisms. This enables the tracking of cells over time and monitoring the progression
of disease states. Unfortunately, this technology is limited to imaging a single cell type at a
time. The ability to image multiple cell types could lead to a greater understanding of
cellular processes, such as the interaction between cancer and immune cells, and could lead
to new therapies. To advance the scope of bioluminescent imaging, we used NanoBit, a split
bioluminescent protein, which produces light when combined in the presence of its small
molecule substrate, furimazine. We cross-screened synthetic halves of NanoBit against a
library of recombinant mutants consisting of mutations at 20 different amino acids sites
near the peptide-protein interface. We selected mutants that bind preferentially to one of
the synthetic peptides to evolve orthogonal pairs of NanoBit. The mutants that were
selected as “hits” were determined by the ratio of luminescence for each peptide. These
“hits” were segregated and pooled for high throughput sequencing. The sequencing results
showed the frequency of how many times a mutant LgBiT was seen for each SmBiT. We
plan to combine the most common mutations to increase binding preferences between the
two peptides. The development of these orthogonal pairs will enhance our ability to
visualize multiple cell types in a single organism.