Difference Between GFP and EGFP

The main difference between GFP and EGFP is that the GFP (stands for Green Fluorescent Protein) is a protein that exhibits bright green fluorescence when exposed to blue light whereas the EGFP (stands for Enhanced Green Fluorescence Protein) exhibits stronger fluorescence than GFP. Furthermore, one other important difference between GFP and EGFP is that the GFP is a wild-type protein isolated from the jellyfish, Aequorea victoria. But, EGFP is an engineered variant of the original wild-type.

GFP and EGFP are two types of proteins that serve as internal chromophores. They do not require any accessory enzymes/substrates, cofactor or gene products to exhibit its color. Therefore, both are used as a reporter of gene expression in molecular biology.

Key Areas Covered

1. What is GFP
     – Definition, Structure, Importance
2. What is EGFP
     – Definition, Structure, Importance
3. What are the Similarities Between GFP and EGFP
     – Outline of Common Features
4. What is the Difference Between GFP and EGFP
     – Comparison of Key Differences

Key Terms

Chromophore, EGFP, GFP, Green Fluorescence, Wild-Type Protein

What is GFP

GFP (green fluorescent protein) is a protein that glows green under blue or UV light. It naturally occurs in the jellyfish, Aequorea Victoria. GFP is made up of 238 amino acids. The size of GFP is 26.9 kDa. GFP is a powerful tool in molecular biology due to its intrinsic fluorescence without any accessory molecule. The fluorescence is because of the covalent rearrangement of contiguous amino acids of the protein. After the folding of the protein, the main chain atoms, Ser65, Tyr66 and Gly67 form the highly conjugated, planar p-hydroxybenzylideneimidazolinone chromophore in the presence of O₂. The crystal structure studies reveal that packing of the chromophore within the core of the β-barrel structure of the molecule protects it from quenching through paramagnetic oxygen, water dipoles or cis-trans isomerization. In addition, the non-covalent interactions of the chromophore with the neighboring molecules enhances its spectral properties.

Figure 1: Ribbon Representation of GFP and Fluorophore

GFP can be introduced into an organism during the transgenic modifications. It can also be maintained through generations. The major drawback of the wild-type GFP is the reduced effectiveness of the protein in cell imaging caused by the low efficiency folding at physiological temperatures such as 37 °C, dropping the fluorescent signal. Also, the maturation rate of GFP inside the host cell is slow while tending to aggregate. Two excitation peaks can be observed due to the presence of two different forms of the chromophore. However, protein engineering has solved most of the problems by introducing variant forms of the wild-type GFP.  

What is EGFP

EGFP (enhanced green fluorescence protein) is a variant of the wild-type GFP with higher-intensity emission with respect to GFP. It is one of the first and most important GFP variants. The two mutations, F64L and S65T, generate EGFP with a greater folding efficiency at 37 °C. Interestingly, EGFP has a single excitation peak at ~490 nm due to the suppressing of the 395 nm peak by S65T since it modulates the ionized state of Glu222 nearby. On the other hand, F64L increases the folding efficiency at 37 °C. Importantly, the codon sequence of EGFP is optimized for the expression in the mammalian cells.

Figure 2: EGFP Expression

Similarities Between GFP and EGFP

• GFP and EGFP are proteins that exhibit bright green fluorescence when exposed to blue light.
• Both can serve as internal chromophore without accessory enzymes/substrates, cofactor or gene products to exhibit its color.
• They form a classical β-barrel fold with the chromophore, containing helix running through the core of the structure. 
• They are used as a reporter of expression in molecular biology.

Difference Between GFP and EGFP

Definition

GFP: A wild-type protein that exhibits green fluorescence under blue or UV light and naturally occurs in the jellyfish, Aequorea Victoria

EGFP: A variant of the wild-type GFP with higher-intensity emission with respect to GFP

Stands for

GFP: Green Fluorescence protein

EGFP: Enhanced green fluorescence protein

Origin

GFP: Wild-type

EGFP: Mutant

64^th Amino Acid

GFP: Phenylalanine

EGFP: Leucine

65^th Amino Acid

GFP: Serine

EGFP: Threonine

Brightness of the Color

GFP: Bright green

EGFP: Brighter green

Excitation Peaks

GFP: Two peaks (395 nm and 490 nm)

EGFP: Single peak (490 nm)

Folding Efficiency at 37 °C

GFP: Low

EGFP: High

Conclusion

GFP is the wild-type protein that exhibits bright green fluorescence when exposed to blue or UV light. EGFP is a variant of GFP that exhibits higher-intensity fluorescence when compared to GFP. Thus, the main difference between GFP and EGFP is the intensity of the green fluorescence each protein emit.

Reference:

1. Arpino, James A. J., et al. “Crystal Structure of Enhanced Green Fluorescent Protein to 1.35 Å Resolution Reveals Alternative Conformations for Glu222.” PLOS Medicine, Public Library of Science, journals.plos.org/plosone/article?id=10.1371/journal.pone.0047132.

Image Courtesy:

1. “Gfp and fluorophore” By Raymond Keller (Raymond Keller (talk)), under auspices of Crystal Protein. – Own work (Public Domain) via Commons Wikimedia 
2. “Fgams ppat egfp puncta” By Zhao A, Tsechansky M, Swaminathan J, Cook L, Ellington AD, et al. (2013) Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies. PLoS ONE 8(2): e56203. doi:10.1371/journal.pone.0056203 (CC BY 3.0) via Commons Wikimedia