Protein Tags
The green fluorescent protein (GFP) is a protein from the jellyfish Aequorea victoria which fluoresces green upon excitation with blue or ultraviolet light. The discovery of green fluorescent protein in the early 1960s by Shimomura has laid the foundation for modern live cell imaging. GFP can be fused to other proteins of interest in a host cell as a noninvasive fluorescent marker in living cells and organisms.
Since then, GFP has been engineered and further developed to produce a plethora of mutants coverving a wide range of colors, including enhanced green, cyan and yellow fluorescent proteins, which exhibit peak emission wavelengths ranging from 425 to 525 nm.
| Protein | Excitation [nm] | Emission [nm] | Extinct. Coeff. [M-1cm-1] | Quantum yield [Φ] | Brightness [nM-1cm-1] | t [0,5] Maturation (37 °C) | Structure | Antibody | |
|---|---|---|---|---|---|---|---|---|---|
| GFP (wt) | 395 | 475 | 509 | 21,000 | 0.77 | 19.8 | 36 min | Dimer | ABIN100085 |
| EGFP | 484 | 507 | 56,000 | 0.6 | 33,6 | 15 min | Weak Dimer | ABIN1169226 | |
| EBFP | 383 | 445 | 29,000 | 0.31 | 6.9 | Weak Dimer | |||
| ECFP | 439 | 476 | 32,500 | 0.4 | 13 | Monomer | |||
| EYFP | 514 | 527 | 83,400 | 0.61 | 44.9 | 9 min | Weak Dimer | ||
Because the construction of red-shifted mutants from the Aequorea victoria jellyfish GFP beyond the yellow spectrum proved largely unsuccessful, researchers have searched for additional options. In 1999 the red fluorescent protein DsRed, derived from Discosoma sea anemones was mentioned first in a publication by Matz et al. Once fully matured, the fluorescence emission spectrum of DsRed features a peak at 583 nm whereas the excitation spectrum has a major peak at 558 nm and a minor peak around 500 nm.
Fluorescent Protein Tags: GFP vs RFP
In comparison to GFP, wild-type DsRed shows certain downfalls but also some unique benefits. Unlike GFP, the fluorescence of DsRed remains stable in acidic pH ranges. Furthermore, DsRed is particulary well suited for fluorescence microscopy, due to its good contrast. Additionally, it has a high quantum yield and is photo stable. On the other hand, maturation of DsRed fluorescence occurs slowly over a time frame of 24 hours. Thus, it cannot be used to monitor proteins with a short halflife. DsRed is an obligate tetramer and can form large protein aggregates in living cells which pose a higher risk for toxicity. Overall, GFP can successfully conjugate with a broader variety of proteins.
RFP Variants
Similar to GFP several RFP variants were engineered to improve its characteristics in general or optimize for specific experiments. DsRed2 contains several mutations at the N-terminus that prevent formation of protein aggregates and reduce toxicity. Red fluorescence emission from DsRed-Express can be observed within an hour after expression, 11 times faster than DsRed. DsRed-Express is a precursor RFP of more recent derivatives including tdTomato and the monomeric mCherry, mOrange, and mStrawberry.
| Protein | Excitation [nm] | Emission [nm] | Extinct. Coeff. [M-1cm-1] | Quantum yield [Φ] | Brightness [nM-1cm-1] | t [0,5] Maturation (37 °C) | Structure | Antibody |
|---|---|---|---|---|---|---|---|---|
| DsRed | 558 | 583 | 75,000 | 0.68 | 49.3 | 26 h 40 min | Tetramer | ABIN129578 |
| DsRed2 | 563 | 582 | 43,800 | 0.55 | 28.6 | 6 h 30 min | Tetramer | ABIN129578 |
| DsRed-Express | 555 | 584 | 38,000 | 0.42 | 12.64 | 42 min | Tetramer | ABIN129578 |
| tdTomato | 554 | 584 | 138,000 | 0.69 | 95.22 | 1 h | Tandem Dimer | ABIN6254170 |
| mCherry | 587 | 610 | 72,000 | 0.22 | 15.8 | 15 min | Monomer | ABIN1440058 |
| mOrange | 548 | 562 | 71,000 | 0.69 | 228 | 2 h 30 min | Monomer | |
| LSSmOrange | 437 | 572 | 52,000 | 0,45 | 23.4 | 2 h 20 min | Monomer |
mCherry is together with mOrange and mStrawberry a member of the mFruits family of monomeric red fluorescent proteins. In comparison to the progenitor DsRed, mOrange, mStrawberry and mCherry produces strong blue- and red-shifted variants. They have a lower molecular weight and will fold faster than tetramers do and therefore disturb the target system less. Out of all of the true monomers developed, mCherry has the longest wavelengths, the highest photostability, fastest maturation and excellent pH resistance. mOrange overcomes mCherry in quantum yield however it does exhibit substantial acid sensitvity. A high extinction coefficient and quantum yield makes the large stoke shift variant LSSmOrange (in combination with T-Sapphire) a suitable candidate for FRET applications.
tdTomato is a genetic fusion of two copies of the dTomato gene which was specifically designed for low aggregation. Its tandem dimer structure plays an important role in the exceptional brightness of tdTomato, which is 5 times higher compared to EGFP. tdTomato's emission wavelength (581 nm) and brightness make it ideal for live animal imaging studies. Because tdTomato forms an intramolecular dimer, it behaves like a monomer and is as photostable as mCherry.
Phototransformable Fluorescent Proteins
Dendra2 is an improved version of the green-to-red photoswitchable fluorescent protein Dendra, derived from octocoral Dendronephthya species. Dendra2 exhibits faster maturation and brighter fluorescence both before and after photoswitching than that of Dendra. In contrast to all other monomeric photoactivatable FPs, which necessarily require UV-violet (e.g., 405 nm laser) light for activation, Dendra and Dendra2 permit the use of blue (e.g., 488 nm laser) activating light. The phototransformative property has allowed highlighting and tracking of subpopulations of cells, organelles, and proteins in living systems. With Dendra2 newly synthesized proteins that are en route to their final destinations can be visualized.
| Protein | Excitation [nm] | Emission [nm] | Extinct. Coeff. [M-1cm-1] | Quantum yield [Φ] | Brightness [nM-1cm-1] | t [0,5] Maturation (37 °C) | Structure | Antibody |
|---|---|---|---|---|---|---|---|---|
| Dendra2 | 490 | 507 | 45,000 | 0.50 | 23 | Monomer | ABIN361314 | |
| 553 | 573 | 35,000 | 0.55 | 19 |
Protein tags are various, mostly short, amino acid sequences that can be used to label proteins. The tag is fused to a recombinant protein using genetic engineering. In general protein tags are used for purification and detection via affinity chromatography, pull-down assays, western blot, immunohistochemistry, fluorescence microscopy, or in live imaging. Due to their structure, tags have different properties and, based on this, different areas of application. Tags are usually engineered onto either the N- or C- terminus in order to minimize tertiary structure disruptions that may alter protein function. Often a protease cleavage site can be inserted between the recombinant protein and the tag to proteolytically dissociate the tag after purification.
What are Affinity Tags?
Affinity tags are attached to proteins to faciliate purification from their biological source using an affinity technique. These include Strep tag and AVI Tag for example which both have a high affinity for biotin. The binding of biotin to streptavidin/avidin is one of the strongest non-covalent interactions known in nature.
The poly(His) tag is a widely used protein tag, which binds to divalent metal ions immobilized on solid phase matrices. His tagged proteins have increased polarity. In alkaline conditions, the histidine residues chelate the metal ion and bind to the carrier.
Fusion Protein Tags
Some affinity tags like maltose binding protein (MBP) or glutathione-S-transferase (GST) are not short AA sequences but large fusion proteins. Beside purification these two have a dual role as a solubilization agent. They ensure proper folding of proteins expressed in chaperone-deficient species such as E. coli and prevent precipitation.
| Tag | Sequence | Matrix | Pros | Cons | Antibody | GST | 211 AA | Glutathione | Supports Folding, Stabilizes Protein, Enzyme Activity Allows Yield Estimate | Large, only Native Purification, only N-Terminal | ABIN1573889 |
|---|---|---|---|---|---|
| MBP | 396 AA | Maltose | Supports Folding | Large, only Native Purification | ABIN103913 |
| HIS | HHHHHH | Ni2+ | Tunable Affinity Resin, Works under Denaturing Conditions | charged, sensitivity (EDTA, DTT, Metalloproteins), Elution Optimization | ABIN1573881 |
| AVI | GLNDIFEAQKIEWHE | Avidin | High Specificity, Gentle Conditions | ABIN1574261 | |
| Strep | WAHPQPGG | Streptavidin | High Specificity, Uncharged, good Purification, one-step Elution | Only Native Purification | ABIN3181089 |
| StrepII | WSHPQFEK | Strep-Tactin | High Specificity, Uncharged, good Purification, one-step Elution | ABIN1573896 |
What are Epitope Tags?
Epitope tags are artificial epitopes, which are cloned in frame with the coding sequence of the protein of interest. Epitope tags are typically composed of amino acid squenences of 10-15 amino acids in length. Due to their relatively small size, they have virtually no effect on the structure of the resulting fusion protein. Most epitope tags are derived from viral genes, which explains their high immunoreactivity. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification. Exapmples are Myc-tag, HA-tag, Spot-tag, or V5-tag.
The DYKDDDDK tag, also known as FLAG® tag is one of the most specific epitope tags artificially created. The FLAG tag's structure was optimized for compatibility with proteins it is attached to. Its hydrophilic nature minimizes effects on function, secretion, or transport of the fusion protein.
| Tag | Sequence | Matrix | Pros | Cons | Antibodies |
|---|---|---|---|---|---|
| HA | YPYDVPDYA | mAb | Low Interference with Protein Structure, Poly-HA Tag Increases Binding Capacity | Cannot Purify Apoptotic Cells | ABIN2443910 |
| Myc | CEQKLISEEDL | mAb | Low Interference with Protein Structure | Can Interfere with Secretory Protein Translocation | ABIN1573879 |
| V5 | GKPIPNPLLGLDST | mAb | Low Interference with Protein Structure | ABIN3181078 | |
| DYKDDDDK | DYKDDDDK | mAb | Low Interference with Protein Structure, high Specificity, good Purification | Limited Binding Capacity | ABIN99294 |
References
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: "Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins." in: Protein expression and purification, Vol. 11, Issue 1, pp. 1-16, (1997) (PubMed).
: "Fluorescent proteins from nonbioluminescent Anthozoa species." in: Nature biotechnology, Vol. 17, Issue 10, pp. 969-73, (1999) (PubMed).
: "Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius." in: Biochemistry, Vol. 45, Issue 21, pp. 6570-80, (2006) (PubMed).
: "Systematic characterization of maturation time of fluorescent proteins in living cells." in: Nature methods, Vol. 15, Issue 1, pp. 47-51, (2019) (PubMed).
: "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein." in: Nature biotechnology, Vol. 22, Issue 12, pp. 1567-72, (2005) (PubMed).
: "Red fluorescent proteins: advanced imaging applications and future design." in: Angewandte Chemie (International ed. in English), Vol. 51, Issue 43, pp. 10724-38, (2013) (PubMed).
: "An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging." in: Journal of the American Chemical Society, Vol. 134, Issue 18, pp. 7913-23, (2012) (PubMed).
: "Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light." in: Nature biotechnology, Vol. 24, Issue 4, pp. 461-5, (2006) (PubMed).
: "Using photoactivatable fluorescent protein Dendra2 to track protein movement." in: BioTechniques, Vol. 42, Issue 5, pp. 553, 555, 557 passim, (2007) (PubMed).
