Green Fluorescent Protein (GFP) has become a transformative tool in biological research, enabling scientists to observe previously hidden processes within living cells. This remarkable protein allows researchers to illuminate specific components, making them visible under a microscope. By rendering cellular structures and activities fluorescent, GFP provides an unprecedented window into the dynamic inner workings of life.
The Origin of GFP
The story of GFP begins with the jellyfish Aequorea victoria. In the 1960s, Osamu Shimomura isolated two proteins from this bioluminescent jellyfish: aequorin and GFP. He found that aequorin emitted blue light in the presence of calcium, and this blue light was then absorbed by GFP, causing it to re-emit green light.
The wider utility of GFP began to be realized in 1992 when Douglas Prasher cloned and sequenced the gene for wild-type GFP. Martin Chalfie subsequently demonstrated in 1994 that GFP could be expressed in other organisms, specifically in the nematode Caenorhabditis elegans, where it successfully illuminated individual cells. Later, Roger Tsien engineered various GFP mutations, creating enhanced versions and a spectrum of different colors. Their combined contributions to GFP’s discovery and development earned them the Nobel Prize in Chemistry in 2008.
The Science Behind GFP’s Glow
GFP’s ability to glow stems from a unique internal structure called a chromophore. This chromophore is formed spontaneously from three amino acids—serine, tyrosine, and glycine—located at positions 65-67 within the protein’s folded structure. When GFP absorbs light, typically in the blue to ultraviolet range, its chromophore becomes excited. This excitation means the chromophore temporarily gains energy.
After absorbing light, the excited chromophore releases this energy by emitting light at a longer, green wavelength. This process is known as fluorescence. A key advantage of GFP is that its chromophore forms and functions without requiring additional enzymes, cofactors, or substrates, other than molecular oxygen. This intrinsic fluorescence makes GFP a versatile tool for biological labeling, as it can glow on its own once produced by a cell.
Attaching GFP to Other Molecules
The utility of GFP in biology comes from its ability to be genetically linked to other proteins. Scientists achieve this by combining the gene that codes for GFP with the gene for a protein of interest. This process involves molecular cloning, where the GFP gene is inserted either upstream or downstream of the target protein’s gene, ensuring they are “in frame” for proper expression.
When the cell produces the protein from this engineered DNA, it creates a “fusion protein” where GFP is physically attached to the protein of interest. Because GFP maintains its fluorescent property when fused, the location and movement of the target protein can be visualized by shining light on the cells. This genetic tagging allows researchers to track proteins without altering their normal function, as GFP generally does not interfere with its fusion partner’s activity or movement.
Unlocking Biological Insights with GFP
GFP tagging makes cellular processes visible in real time within living systems. Researchers can track protein localization, observing where they reside within a cell, such as in the nucleus, cytoplasm, or specific organelles like mitochondria. This allows scientists to monitor how proteins move and interact over time, providing insights into cellular activities.
Beyond protein tracking, GFP is used to monitor gene expression, revealing when and where a specific gene is active within a cell or organism. This helps researchers understand gene regulation. GFP also enables the visualization of cellular structures and developmental processes in living organisms, from single cells to entire embryos. The non-invasive nature of GFP tagging means observations can occur without harming cells, a key advantage over older techniques that often required fixed, non-living samples.
Broadening the Scope of GFP Research
The initial GFP led to a diverse palette of fluorescent proteins. Scientists engineered GFP variants that emit different colors, including blue, cyan, yellow, and red. This array allows researchers to tag multiple proteins or cellular components simultaneously, observing their interactions and relative positions.
The versatility of GFP and its colorful relatives extends beyond basic research. These fluorescent tools are employed in drug discovery to screen for compounds affecting cellular pathways. They also find use in environmental monitoring, such as engineering bacteria to glow in the presence of pollutants.