Functional Fusion Proteins by Random Transposon‐Based GFP Insertion

Abstract

Fusions with fluorescent proteins are usually created by fusing the ends of two coding sequences. Appending the coding region of a fluorescent protein to the N- or C-terminus of another protein is typically the easiest way of creating a functional, fluorescent fusion protein. Another strategy involves placing the fluorescent protein in the middle of another protein. Such sandwich fusions are feasible, and there are many reasons for creating these fusion proteins. For example, sandwich fusions can be used to place two fluorescent proteins close to one another for optimization of a biosensor based on fluorescence resonance energy transfer, or they can be used to place the fluorescent protein in a region that moves during conformational changes of the host protein. Designing a sandwich fusion that produces a functional, fluorescent fusion protein is often challenging. This protocol describes an alternative approach. A simple, in vitro, transposon reaction is used to randomly insert the sequence encoding a fluorescent fusion protein into a target protein. This random labeling strategy makes it possible to create a small library of sandwich fusion proteins that can then be screened for activity. The approach makes it possible to test many possible solutions to the complex problem of building new biosensors.