Proteins that possess a membrane-translocating sequence (MTS) derived from the FGF signal sequence can be translocated into cells (Rojas et. al, 1998). The MTS is proposed here for use in developing high-throughput (HTS) and ultra high-throughput drug screening (UHTS) assays. Increasingly, assays used in HTS and UHTS are fluorescence-based due to their sensitivity. It would be a significant breakthrough in drug discovery if one could simply add a fluorescent reporter protein or peptide into cells, to any desired final concentration, to assess the activity of a molecular target. We propose to label MTS-containing recombinant proteins of interest with fluorescent probes and then determine whether these proteins can be translocated into cells and report various specific events. Use of the dye-labeled MTS-containing proteins as a fluorescent reporter for intracellular events will depend on a number of parameters that will be assessed. These parameters include optimal accessibility to various locations within the cell, functionality of the translocated protein, and translocation efficiency that results in a homogeneous fluorescent signal from cells that is compatible with HTS/UHTS instrumentation (e.g., plate readers). We also plan to optimize the rate of translocation into various cells by identifying key residues within and flanking the MTS, and by altering loading conditions.
There are currently very few fluorescent reporters available for cell-based assays in HTS/UHTS, and these reporters are limited in their range of use. The MTS approach for introducing fluorescent reporters into living cells will potentially enable researchers to have nearly the same flexibility for doing cell-based assays as is currently available for doing biochemical assays. Thus, this technology has potentially broad applications for both the pharmaceutical and biotechnology industries.
