Green Fluorescent Protein

Green Fluorescent Protein (GFP) is a naturally occurring fluorescent molecule originally isolated from the jellyfish Aequorea victoria. It emits a bright green light when exposed to ultraviolet or blue light, making it one of the most significant discoveries in modern molecular biology. GFP has revolutionised research in genetics, cell biology, and biochemistry by enabling scientists to visualise and track molecular processes in living cells in real time without disrupting their normal function.

Discovery and Origin

The story of GFP began in the early 1960s when Japanese marine biologist Osamu Shimomura and his team isolated two proteins—aequorin and GFP—from the jellyfish Aequorea victoria, collected from the waters near Friday Harbor in Washington, USA. Aequorin emitted blue light upon binding with calcium ions, while GFP absorbed this blue light and re-emitted it as green fluorescence.
It was only in the 1990s that GFP’s full potential as a molecular tool was realised. Douglas Prasher first cloned the gene encoding GFP, and Martin Chalfie demonstrated its expression in living organisms, showing that the protein fluoresces without any external additives. Roger Tsien later enhanced the protein’s brightness and colour range through mutagenesis, leading to the development of variants emitting different colours.
The groundbreaking impact of this work led to Shimomura, Chalfie, and Tsien jointly receiving the 2008 Nobel Prize in Chemistry for their contributions to the discovery and development of GFP.

Structure and Molecular Properties

GFP is a globular protein consisting of 238 amino acids with a molecular weight of approximately 27 kilodaltons (kDa). Its structure resembles a beta-barrel, composed of 11 beta-sheets surrounding a central alpha-helix.
At the centre of this barrel lies the chromophore, the light-emitting component of the protein, which forms spontaneously through a chemical reaction involving three specific amino acids: serine (Ser65), tyrosine (Tyr66), and glycine (Gly67).
When excited by ultraviolet or blue light (at about 395 nm or 475 nm), the chromophore emits a bright green fluorescence at 509 nm. This intrinsic fluorescence requires no additional cofactors, enzymes, or substrates, making GFP uniquely self-sufficient for use in living cells.

Variants and Engineering

Through genetic engineering, researchers have created numerous GFP variants with altered excitation and emission properties, improved stability, and enhanced brightness.
Important variants include:

• Enhanced GFP (EGFP): Modified to increase fluorescence intensity and stability.
• Blue Fluorescent Protein (BFP) and Cyan Fluorescent Protein (CFP): Created by altering amino acids in the chromophore region to shift emission colour.
• Yellow Fluorescent Protein (YFP): Emits yellow light, useful for multi-colour imaging.
• Superfolder GFP (sfGFP): Designed to fold correctly even under harsh cellular conditions.

These variants have allowed scientists to label multiple proteins simultaneously, enabling detailed observation of cellular processes through multi-colour fluorescence microscopy.

Principle of GFP Function

The functional principle of GFP lies in its ability to act as a reporter molecule. When the gene encoding GFP is inserted into an organism’s DNA and linked to another gene of interest, GFP is expressed alongside the target protein. The fluorescence emitted by GFP then allows researchers to:

• Visualise when and where the target gene is active.
• Track the location and movement of proteins within living cells.
• Measure gene expression levels in real time.

This capability to non-invasively monitor cellular activity has made GFP a cornerstone of modern biological imaging.

Applications in Scientific Research

GFP and its derivatives are widely used in diverse areas of biological and biomedical research:
1. Cell Biology: GFP is used to study protein localisation, cell structure, and intracellular transport. By tagging specific proteins, scientists can observe processes such as mitosis, endocytosis, and apoptosis under live-cell imaging systems.
2. Developmental Biology: By introducing GFP into embryos, researchers can track tissue differentiation, organ formation, and gene expression patterns during development in organisms such as zebrafish, fruit flies, and mice.
3. Neuroscience: GFP helps visualise neural networks and trace connections between neurons. Specialised variants like Synapto-pHluorin report synaptic activity in real time.
4. Molecular Genetics: GFP serves as a reporter gene, providing a visual marker for successful gene insertion or promoter activation in genetic engineering experiments.
5. Medical and Pharmaceutical Research: GFP-based systems are employed to monitor the effects of drugs on cellular pathways, cancer cell metastasis, and pathogen-host interactions.
6. Environmental Monitoring: Genetically engineered microorganisms expressing GFP are used to detect pollutants or monitor the presence of toxins in ecosystems.
The ability to visualise biological processes at the molecular level has made GFP indispensable in both basic and applied sciences.

Advantages of GFP

• Non-toxic and self-sufficient: Requires no external substrates or cofactors.
• Real-time observation: Enables live imaging of dynamic biological processes.
• Stable and reproducible: Can be expressed in a wide variety of organisms, from bacteria to mammals.
• Multi-colour flexibility: Different GFP derivatives allow simultaneous tracking of multiple targets.
• Quantitative tool: Fluorescence intensity can be measured to quantify gene or protein activity.

These advantages have transformed GFP into one of the most versatile tools in molecular and cellular biology.

Limitations and Challenges

Despite its success, GFP use is associated with certain limitations:

• Photobleaching: Prolonged exposure to light reduces fluorescence intensity.
• Maturation time: The chromophore requires time to develop, causing delays in signal detection.
• Protein misfolding: In some cases, fusion proteins may not fold correctly, affecting normal cellular function.
• pH sensitivity: Fluorescence can diminish under acidic conditions.
• Size constraints: The relatively large size of GFP may interfere with the function of the target protein when fused.

To overcome these challenges, researchers continue to develop modified GFP variants with faster maturation, higher stability, and reduced interference with host proteins.

GFP and the Fluorescent Protein Revolution

The discovery of GFP sparked a revolution in biological imaging, inspiring the development of a family of fluorescent proteins from other marine organisms, such as the Red Fluorescent Protein (RFP) from Discosoma and mCherry, a popular red variant. Together, these proteins allow scientists to study complex cellular interactions through techniques like fluorescence resonance energy transfer (FRET) and super-resolution microscopy.
In addition, GFP has been instrumental in the creation of biosensors—engineered proteins that emit light in response to specific cellular events such as calcium flux, pH change, or enzyme activity. These tools have enabled the study of molecular signalling pathways in unprecedented detail.

Legacy and Scientific Impact

The development of GFP fundamentally transformed life sciences, providing a window into the microscopic world of living systems. Its introduction shifted biological research from static to dynamic observation, allowing real-time visualisation of molecular events inside living organisms.
The simplicity, versatility, and universality of GFP technology have made it a cornerstone of 21st-century biology. Today, its principles are applied in genome editing, synthetic biology, drug development, and regenerative medicine, continuing to drive scientific innovation.