GFPfusies

GFPfusies, also known as GFP fusions, are fusion proteins in which green fluorescent protein (GFP) is genetically attached to another protein of interest to visualize its localization and dynamics in living cells. GFP originates from the jellyfish Aequorea victoria and emits green fluorescence when excited by blue light, enabling noninvasive imaging without added substrates.

To create a GFP fusion, the GFP coding sequence is inserted in frame with the target protein’s coding sequence, commonly at the N- or C- terminus. Flexible linkers can reduce steric interference and preserve function. The tag’s position, linker length, and expression level are chosen based on the biology of the protein. Expression systems include bacteria, yeast, plants, and mammalian cells, using transient transfection or stable integration.

Applications include determining subcellular localization, monitoring protein trafficking and turnover, and enabling live-cell time-lapse imaging. GFP fusions support co-localization studies with organelle markers and can be used in conjunction with other fluorescent reporters for multicolor imaging.

Limitations and considerations include potential disruption of protein function or localization due to the tag, increased molecular size, maturation time, and photostability. Overexpression can cause artifacts. To mitigate these issues, researchers test multiple tag placements, use monomeric GFP variants, and verify that fusion proteins retain native activity.

Variants and extensions include monomeric GFP derivatives (such as sfGFP, mEGFP) and other spectral proteins for multi-color experiments; advanced approaches also include split-GFP or FRET-based methods that rely on GFP components but extend beyond simple fusions.