Constructing and expressing fluorescent protein fusions

Fluorescent protein fusions (FPFs) have been used to address a wide range of questions in individual cells as well as in specific tissues of a particular organism. However, investigators must take extreme care when using FPFs to ensure that the resultant fusion protein is expressed at or close to the endogenous level of the parent protein, and also that it is full length, localizes correctly, and behaves normally once incorporated in the cell. Because the molecular mass of the fluorescent protein (FP) itself is 27 kDa, one must consider the potential effects of placing such a large tag in association with a protein under investigation. This article discusses how these goals can be achieved and provides examples to assist the investigator in designing and implementing experiments using FPFs.