CUT-AND-PASTE GFP

Abstract

A NEW IN VITRO METHOD for splitting and reassembling green fluorescent protein (GFP) gives scientists synthetic control of the composition of this common fluorescent tag. The method will not only help scientists better understand the workings of GFP but also inspire the design of improved proteins for fluorescent labeling in cells. GFP consists of β-strands that form a barrel and a chromophore-containing α-helix inside the barrel. In the new method, GFP is cut at predetermined sites, and to reassemble the protein, the parts removed by the cuts are replaced with synthetic components. The method was inspired by so-called split GFP assays, which are widely used to detect protein interactions in vivo. In these assays, GFP is expressed as two pieces, each fused to proteins whose interaction is being probed in cells. When the proteins interact, GFP forms from the two pieces, and the assembly fluoresces. Although studying the properties of these smaller pieces of GFP ...