| In order to observe biological processes, such as protein-protein interactions, it is desirable to link the biological process to the expression of some easily detectible reporter protein. Current flow cytometry technology allows for the detection of fluorescent protein expression on a cell by cell basis, at the rate of thousands per second. This makes fluorescent proteins a logical choice as a reporter. In this work, green fluorescent protein (GFP) is used to (1) report on the binding of a zinc finger protein to a target fragment of DNA, and to (2) attempt to create a molecular sensor through the insertion of GFP into maltose binding protein. Zinc finger binding was linked to the expression of GFP through the modification of a bacterial one-hybrid system developed by Hochschild and coworkers. This system was then used to isolate, from a library of randomized zinc fingers, those proteins that are capable of binding the target site. Unlike the zinc finger binding detection system, the mechanism used to link maltose binding to cell fluorescence does not involve binding-dependent expression of the fluorescent protein. Instead, our goal was to create a molecular switch, in which the fluorescence of the protein is dependent upon ligand binding. Combinatorial protein engineering was used to construct libraries of fusion proteins where a form of GFP is inserted randomly into maltose-binding protein. Cells expressing fluorescent library members were isolated through Fluorescence-Activated Cell Sorting (FACS) and these proteins were screened to determine if the fluorescence is impacted by presence of absence of maltose. |
