Enhanced green fluorescent protein (EGFP) is a modified protein that acts as a biological highlighter, allowing scientists to illuminate specific components within living cells. It is derived from a naturally occurring protein but altered to improve its utility in research. By linking the gene that produces EGFP to other genes of interest, researchers can visually track cellular processes. The protein forms an internal light-emitting element that requires only oxygen to function, making it a self-sufficient marker.
The Original Glowing Protein
The origin of this molecular light is the crystal jellyfish, Aequorea victoria. In the 1960s, researcher Osamu Shimomura was studying bioluminescence in these jellyfish when he discovered a protein that glowed green under ultraviolet light. This was the first identification of what became known as green fluorescent protein, or GFP.
For decades, its potential remained unrealized until the 1990s, when Martin Chalfie and Roger Y. Tsien demonstrated its utility. Chalfie showed that the gene for GFP could be expressed in other organisms, acting as a luminous tag. Tsien later expanded on this by deciphering its structure and creating a palette of fluorescent proteins of different colors. The work of Shimomura, Chalfie, and Tsien led to their receiving the Nobel Prize in Chemistry in 2008.
The original, or wild-type, GFP had limitations that restricted its use. It was not intensely fluorescent and tended to form non-functional clumps when produced. A more significant issue was its failure to fold into its correct, light-emitting shape efficiently at the warmer temperatures found in mammals (37°C), making it difficult to use in human cells.
Creating a Brighter Beacon
The development of EGFP was a direct response to the shortcomings of its natural predecessor. Scientists engineered a version suitable for mammalian systems by making precise changes to the protein’s genetic code. The goal was to create a protein that was brighter, more stable, and produced more reliably.
A breakthrough in 1995 was the S65T mutation, a single amino acid substitution that improved the protein’s spectral properties. This change simplified how the protein absorbed light and increased its fluorescence. Shortly after, the F64L mutation was introduced, which helped the protein fold correctly at the 37°C temperature of mammalian cells.
These amino acid changes resulted in a protein approximately 35 times brighter that matured much faster than the original version. Scientists also optimized the genetic instructions for building the protein through codon optimization. This process alters the DNA sequence to better match mammalian cells, leading to more efficient production of the final EGFP product.
How EGFP Works as a Biological Lightbulb
EGFP’s ability to glow is based on fluorescence. Similar to a highlighter pen, it absorbs light of one color—blue—and then emits it as green light at a slightly longer wavelength. This transformation is performed by a chemical structure at the protein’s core.
This light-emitting center is called a chromophore. It forms spontaneously from three of the protein’s own amino acids in a chemical reaction that requires only the presence of oxygen. This self-contained mechanism means EGFP does not need other cellular components or cofactors to function.
The chromophore is protected by the protein’s overall structure, a shape known as a beta-barrel. This structure consists of protein strands that wrap around to form a cylinder, creating a protective shield for the chromophore inside. This architecture safeguards the light-emitting machinery and helps it maintain the configuration needed for fluorescence.
Applications in Scientific Discovery
Scientists use EGFP in two primary ways: as a reporter gene and as a protein tag. When used as a reporter, the EGFP gene is placed under the control of the same genetic “on-switch” as a gene of interest. When the cell activates the target gene, it also produces EGFP, causing the cell to glow green and reporting when and where that gene is active.
The second application is using EGFP as a molecular tag to track the location and movement of other proteins. The gene for EGFP is fused directly to the gene of a target protein, creating a hybrid that carries the fluorescent marker. Researchers can then use a microscope to watch the tagged protein in real time, revealing its journey through cellular compartments.
These applications have advanced many fields of biology and medicine. Neuroscientists have used EGFP to watch individual neurons grow and form connections in a living brain. Cancer researchers tag cancer cells with EGFP to observe metastasis, tracking how tumor cells spread. Virologists also use it to monitor a viral infection as it moves from cell to cell.