Protein complementation assays (PCAs) are routinely used to probe the biochemical nature of protein interactions in live cells and animals. Fully genetic (i.e., no exogenous substrate required) PCA systems based on the reconstitution of split fluorescent proteins provide high contrast, but lack the sensitivity enhancement provided by enzymatic amplification. PCA systems based on split bioluminescent enzymes can provide high contrast but require the supplementation of luciferin substrate. Likewise, the split beta-galactosidase and split beta-lactamase reporter assays require supplementation with synthetic fluorogenic substrates. To circumvent the need for addition of an exogenous substrate, we are pursuing the development of a new PCA system based on a split enzyme that employs an endogenous substrate to produce a fluorescent molecule. This enzyme is an S-Adenosylmethionine dependent Urogen Methyltransferase (CobA), the first enzyme from the Vitamin B12 (Cobalamin) pathway, and its substrate is a porphyrin intermediate in the heme pathway. Thus a CobA based PCA could produce high fluorescent contrast while being effective in all animals, plants, and organisms that synthesize heme (or chlorophyll).

Towards a fluorogenic enzyme complementation assay that uses an endogenous substrate

251st National Meeting (2016)

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