The purified tag is a known protein, or peptide, which can be fused into the protein you are interested in. Because these tags are easy to identify, there are many antibodies to choose from and they are very easy to detect. The method of labeling is mainly recombinant DNA, that is, the coding DNA carrying protein you are interested in is integrated with the labeling, and the plasmid is transferred into the cell. When the plasmid is expressed, the fusion tag will be attached to your protein.
The selection of fusion label is very important, and so is the selection of joint sequence. The connector sequence connects your protein to the label to ensure that the protein can be folded correctly and function normally.
Why use fusion tags?
Advantages:
(1) isolated protein even if there is no specific antibody against the protein.
(2) After purification, the fusion label needs to be cut off if necessary.
(3) Multiple markers can be attached to the same protein for various purposes.
(4) Avoid antibody interference during immunoprecipitation.
(5) Fluorescent labeling can be used to observe protein in living cells.
Disadvantages:
(1) Some labels will affect the function of protein.
(2) Need to optimize the label position.
This paper only introduces all kinds of labels, and can't say which label must be placed in which position, which depends entirely on your experimental needs.
If you are not sure where your tag is better, you need to try several schemes, such as changing or adding a protease site, modifying the linker sequence, or adding multiple tags for different purposes.
At present, there are three main types of fusion tags:
Epitope tag: generally short peptide sequence, which can be used in immune technology applications, such as WB and Co-IP.
Affinity label: generally long, it can be used for protein purification or increasing the solubility of protein.
Fluorescent labeling: It can be used for living cells/dead cells, and is generally used for imaging experiments, such as cell localization and * * * expression experiments.
It all depends on what you want to study, protein: How does protein fold? Which end is functional? For example, if the C-end is folded inside the protein, you can't detect the signal of the fusion tag; Or your protein will be cut off at one end after translation, so will your label.
If you don't know how to fold your protein, it's best to try both the C end and the N end when you do it for the first time. It is reported that the C- terminal fusion protein is more located in the expected cell region than the N- terminal labeled fusion protein. But this is not entirely the case.
Not all labels are the same: each label has its own uses and limitations. For example, you need to make your protein more soluble and use labels to purify it. Tandem affinity purification (TAP). TAP originally refers to the fusion of a series of specific domains into protein: calmodulin binding peptide (CBP) and protein A(ProtA) of Staphylococcus aureus, which are separated by the cleavage site of tobacco etching virus (TEV). However, this technology can now be used to fuse 2-3 fusion tags to protein, but usually it still contains the original TAP components. These labels can be integrated into one or both ends of the protein. If the labels need to be cut off after use, it is usually recommended to connect the labels in series.
His label is widely used in TAP because it is very small and very effective for purification experiments. Usually used with maltose binding protein (MBP) to increase solubility. Another commonly used combination is GFP and His.
TAP tag has many advantages, but it is very bulky, which may destroy the function of protein. In addition, if you use a label containing CBP, it may interfere with the calcium signal. So, put as few labels as possible on your protein.
Some small labels have little effect on protein's function, but some big labels may have unexpected effects. In this case, you usually need to cut off the label after testing. This requires the use of proteases or peptides at specific sites.
Site-specific protease will not affect your protein function in theory. However, proteases are usually removed after cleavage. One solution is to fuse the protease with the tag and remove it by affinity chromatography. The choice of several protease recognition sites depends on your needs. One thing to note is, is the fusion label at the N-end or the C-end? Cutting off the label from the N-terminal will leave fewer residues, while cutting off the label from the C-terminal will leave more than 4-6 residues on your protein. The following is a list of several commonly used protease sites (English version, please check it yourself if necessary):
Affinity tags are so named because they are often used for affinity purification. Affinity purification is a technique to purify protein from cell lysate. See the figure below:
GST is a protein composed of 2 1 1 amino acids, and its molecular size is about 26KD. Natural GST can protect cells from harmful components and oxidative stress. GST is characterized by its affinity for tripeptide and glutathione, and can be used for affinity chromatography. When a solution containing GST passes through a column on which glutathione is immobilized, GST binds to glutathione and is separated from the solution. After binding, it can be eluted with 10mM glutathione.
Precautions:
(1) Pollution: Heat shock proteins are sometimes eluted by GST. You can see this pollution through SDS-PAGE glue. In order to remove the pollution of heat shock protein, the cell lysate can be treated with 5mM calcium chloride and 5mM ATP before purification.
(2) Loss of protein function: Because GST is a big label, it usually exists in solution in the form of dimer. It may reduce your protein activity and function. Cleavage with protease (e.g. thrombin, factor Xa or pre-cleavage) can be used.
PolyHis tag is widely used in protein purification because of its small size and stable binding. Although His tag can have 2- 10 histidine residues, the 6×His tag, or hexatag, is most commonly used. Histidine forms coordination bonds with immobilized transition metal ions, which can be used for protein purification. Cobalt and zinc columns can be used for fixed metal affinity chromatography (IMAC), but nickel columns are usually used. Because His tags are short peptide sequences, the optimal position of the tags depends on protein specificity, and they rarely affect the properties of the fusion protein.
Precautions:
(1) Endogenous His: His residues exist widely in mammals and insects, so it has a high background signal. Washing with 5- 10mM imidazole can avoid background combination, but it will wash off your protein prematurely. In addition, attention should also be paid to background binding when detecting with His antibody.
(2) It is not recommended to fuse protein with metal center to His-tag, because metal may be adsorbed by affinity resin.
(3) Reducing agents, such as dithiothreitol (DTT) or β-mercaptoethanol, should be avoided because they will reduce the affinity of the resin.
Biotin, also known as vitamin H, is a small molecule with a molecular weight of 244Da. It is usually attached to the secondary antibody as a visualization technique for ELISA and WB. Biotin can form a very strong combination with streptavidin and avidin molecules, which can be used for affinity purification. Because biotin is a small molecule, it will not affect the function of protein. Another valuable label is the Streptococcus label. Because its length is 8 amino acids (WRHPQFGG), it will not affect the function of protein, and it can bind with biotin reversibly in the same pocket. This means that protein fused with Strep-tag can be effectively purified by using strptavidin resin and eluted with biotin under mild buffer conditions.
Precautions:
(1) Affinity resin of tetramer and monomer: Biotin coated column can be used to purify biotin-labeled protein, and protein can be used in tetramer or monomer form. The elution process of tetramer abidin chromatographic column needs strong denaturant, such as urea or guanidine hydrochloride, and the monomer resin can be gently eluted with 10mM biotin buffer.
(2) The mammalian system contains at least four biotinylated protein, which will be eluted with protein of interest. However, nonspecific binding is rarely encountered in the E.coli system.
Epitopes are part of antigens and recognized by antibodies. Therefore, epitope tags are usually used in antibody-based analytical experiments. Epitope tags are generally shorter than affinity tags, so they rarely affect the function of protein. Although they can also be used for affinity purification, columns based on immune antibodies are expensive and not as effective as affinity tags. Epitope tags have great advantages in detection. Widely used in cell culture and immunoprecipitation.
The expression level of human c-myc in proliferating cells is low, which plays an important role in human tumorigenesis. C-myc tag comes from the C-terminal of C-myc gene and can be effectively recognized by antibodies. Therefore, it is widely used in protein detection, such as WB, immunoprecipitation and flow cytometry.
Precautions:
(1) Fusion into secretory signal: Although the c-myc tag can be placed at the N-terminal or C-terminal of protein, it is not recommended to be fused into secretory signal pathway, which will affect the localization of secretory signal pathway.
(2) Affinity purification: Although it can also be used for protein purification, it is rarely used. Because elution needs to be carried out at a very low pH value, which will affect the function of protein.
Human influenza virus hemagglutinin (HA) tag is derived from HA glycoprotein, which exists on the surface of influenza virus and is responsible for the infectivity of the virus. As HA is a small peptide tag, it basically does not affect the function of protein, and can be used for protein detection, such as ELISA, WB, immunoprecipitation, etc. Anti-HA antibody can also be used for purification of protein.
Precautions:
Affinity purification: HA tag is not recommended to label protein in apoptotic cells. HA tag will be cut by caspase 3 and caspase 7, resulting in decreased immune reactivity.
DDDK or FLAG is the only patent tag (sigma), which is more hydrophilic than other epitope tags. If necessary, the enterokinase cleavage sequence contained in the DDDK tag can cleave the tag from the fusion protein.
Precautions:
Affinity purification: Although anti-DDDK antibody can effectively purify protein, it is usually more expensive than other chromatographic columns.
The V5 tag is derived from P protein and V protein in paramyxovirus simian virus. V5 tags come in two sizes, 9- 14 amino acids. But the longer ones are commonly used. Sometimes V5 tags are used in combination with His- tags.
It should be noted that:
Cross-reaction: If the mammalian expression system is used, cross-reaction may occur.
Since 1992, GFP gene has been cloned, and there are many fluorescent tags available at present. One of the biggest advantages of fluorescent labeling is that it is nontoxic, so it can be used in living cells. Although the fluorescent label is large, it has little effect on most protein functions.
Although GFP is a fluorescent marker with a size of 26.9KD, there are many other markers available.
If you plan to use a variety of fluorescent markers, it is very important to choose different excitation and emission peaks. If the emission peaks overlap, it is difficult to distinguish the two kinds of fluorescence.
Usually, you want to label the protein with the brightest fluorescent marker in the available spectrum, so that you can get a clear signal and overcome any potential background fluorescence. The brightness value is the product of protein's extinction coefficient and quantum yield. However, the figures obtained may be difficult to interpret. Therefore, a common measurement method is to compare the brightness of fluorescent labels with standard labels (such as EGFP).
Maturity defines the time when fluorescent labels fold correctly, form chromophores and start to emit fluorescence. In time-sensitive events of living cells, short maturation time is very important. For example, SfGFP, 10 minutes can be folded, while mOrange usually takes 4 hours.
Bleaching is a measure of photostability. That is, how long after the chromophore is excited, it loses its luminous ability. If you are going to do a long delay experiment, consider a label with high light stability. The bleaching half-life of T- sapphire is 25 seconds, while EGFP is much more stable, generally 174 seconds.
Like most protein, fluorescent labeling is influenced by ph value, temperature and oxygen level. Depending on the conditions you use, you may need to choose the correct label.
The pH value will affect the excitation and emission peaks, and most fluorescent labels are sensitive to acid. Some can even change the fluorescence intensity when the pH value changes (for example, pHTomato). PKa value is a good indicator of pH sensitivity.
In addition, both temperature and oxygen level will affect the ripening time. Hypoxia usually delays maturation time. However, the new fluorescent marker UnaG can emit fluorescence even under the condition of low oxygen level.
Because most fluorescent markers come from jellyfish or coral proteins, rather than mammalian cells or tissues, there are species differences in amino acid codons that may be used, which may lead to low expression level and weak signal.
Fortunately, many new versions of fluorescent labels have been codon-optimized for high expression in mammals. So before using it, you need to confirm whether your sequence can be expressed in the target system.
Another important point is to determine whether your label is monomer or dimer (monomer is usually represented by "m", such as mCherry). This will affect your experiment. Many early fluorescent markers are easy to form oligomers, which may affect the function of your fusion protein. For example, EGFP is a monomer label, but at high concentration, it can form dimer, destroy subcellular organelles or destroy experiments such as FRET.