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How to Select Protein Tags

You may often face the problem of choosing protein tags. PackGene provides you with a reference for choosing protein tags below.

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Tag Names Introduction to protein tags


They are enhanced green fluorescent protein (eGFP), enhanced yellow-green fluorescent protein (eCFP), enhanced yellow-green fluorescent protein (eYFP), monomer red fluorescent protein (mCherry), with different excitation wavelengths and emission wavelengths, both It is derived from wild-type fluorescent protein through amino acid mutation and codon optimization. As far as eGFP is concerned, compared to GFP, its fluorescence intensity is stronger and its fluorescence properties are more stable. At the same time, the Kozak sequence constructed in the vector makes the eGFP-containing fusion protein expressed more efficiently in the eukaryotic expression system. mCherry is a monomer red fluorescent protein with the best performance evolved from DsRed. It can be shared with GFP series fluorescent proteins to achieve multi-color labeling. In vivo and in vitro experiments have shown that when mCherry is fused with foreign proteins at the N-terminus and C-terminus. The activity of the protein and the function of the target protein being fused have no significant influence on each other.



The luciferin derived from living organisms, the common ones are firefly luciferase, Renilla luciferase and Guassia luciferase. Luciferase is used as a “reporter protein” in reporter gene detection method or luciferase assay (Luciferase Assay). Unlike ordinary fusion protein tags, reporter genes constructed with luciferase can be used for quantitative analysis of target genes. Therefore, it is often used to study the function and regulation of promoters and miRNA 3’UTR clones, because their regulation of the target gene can be gradual, rather than simply on and off.
HA The HA tag (amino acid sequence: YPYDVPDYA) is a short sequence derived from amino acids 98 to 106 of the human influenza virus hemagglutinin (HA) protein, with a molecular weight of 1.1 KDa. It is currently one of the widely used epitope tags and can form Strong antibody recognition site. After the HA tag is fused to the C-terminus or N-terminus of the target protein by molecular biological means, the anti-HA tag antibody can be used for the detection, separation and purification of the HA-labeled target protein without the need for protein-specific antibodies or probes.
Flag Flag fusion tag is a short peptide composed of eight amino acids (DYKDDDDK), specially designed for immunoadsorption purification of recombinant proteins. The Flag tag has a short structure and contains an enterokinase cleavage site between it and the fusion protein, which can be removed by enterokinase cleavage.
His6 6xHis tag, also known as polyhistidine tag, His6 tag or hexahistidine tag, is a type of target protein in the transfected cell linked by at least 6 histidine residues on the C-terminus or N-terminus Consists of the amino acid sequence. Relatively small molecular weight, low immunogenicity, hydrophilicity and variability in the presence of detergents and other additives, the sequence of histidine residues can be bound to several types under specific buffer conditions Fixed ions (such as nickel, cobalt and copper) to achieve the purpose of easy detection and purification of His tag protein. Therefore, under natural and denatured conditions, the polyhistidine tag is recognized as the most widely used affinity tag for a series of protein purification purposes.
GST Glutathione S-transferase (GST) is one of the first epitope tags to be used; it can be placed at the N- or C-terminus and can express protein soluble. Glutathione binding resin was used for purification.
HSV Herpes simplex virus (HSV) is derived from glycoprotein D precursor envelope protein and is short (QPELAPEDPED), so it is unlikely to interfere with protein structure or function.
MBP Maltose binding protein (MBP), the size of the tag (43kDa) helps it increase the production of soluble protein in E. coli. Together with GST and thioredoxin, it is also very popular as a solubilizing label. Use low-cost amylose resin (Asn10 spacer between the label and the protein can enhance the binding ability with the resin) to achieve purification.
c-Myc C-Myc tag protein is a small tag containing 11 amino acids. The tag sequence is Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu. These 11 amino acids are expressed as epitopes in different The corresponding antibody can still be recognized in the protein framework. C-Myc tag has been successfully applied in Western-blot hybridization technology, immunoprecipitation and flow cytometry, and can be used to detect the expression of recombinant proteins in target cells.
T7 This is a 260-residue tag from the gene 10 product of T7 phage; it can enhance the expression level in E. coli.
Snap SNAP-Tag is obtained from human O6-methylguanine-DNA-alkyltransferase. The active sulfhydryl site of SNAP accepts the side chain benzyl group carried by benzyl guanine, releasing guanine. This new thioether bond covalently binds the target protein carried by SNAP to carry the label carried by the benzyl group. Biotin or fluorescent substrates of various colors (such as fluorescein, rhodamine) can penetrate into cells, which can be used to conveniently and quickly label and detect SNAP-Tag fusion proteins in living cells. It can also specifically label E. coli , SNAP-tag fusion protein in cell extracts of yeast and mammals or purified protein.
SV40 NLS The nuclear localization sequence PKKKRKV or PKKKRKVG, the molecular weight is 883.13, the wild-type amino acid sequence is Pro-Lys-Lys-Lys-Arg-Lys-Val, even if a single amino acid mutation produces a mutant NLS (Pro-Lys-Tyr-Lys- Arg-Lys-Val) can also prevent this protein from entering the nucleus and staying in the cytoplasm.
P2A/T2A The 2A peptide was found in the Foot-and-Mouth Disease Virus (FMDV). It is a small “self-cleaving” peptide identified from it, with an average length of 18-22 amino acids. 2Apeptide “simultaneous transcription and simultaneous translation”
IRES The IRES element has the function of independently recruiting ribosomes without relying on the cap structure, and in turn can initiate the translation of downstream genes. IRES “simultaneous transcription, independent translation”
V5 Derived from amino acids 95-108 of the P/V protein of Paramyxovirus SV5 (GKPIPNPLLGLDST), the V5 tag is often fused and expressed on the N-terminus or C-terminus of the target protein for analysis and identification of the target protein.
dTomato Among all the spectral types, the brightest fluorescent protein is the tandem dimer Tomato (dimeric Tomato, dTomato). The tandem dimer contains 2 copies of dTomato linked by a linker of 12 amino acid residues. Due to a pair of chromophores, dTomato is extremely bright and has unique light stability. The biggest disadvantage of dTomato is its large molecular weight, which can interfere with the folding of the fusion protein in some cases.
CAT Chloramphenicol acetyltransferase (CAT), this 24kDa tag is also used as a reporter gene, and it retains its activity when fused with most proteins. This means that it can be used to directly measure expression levels without the need for PAGE or immunoassay.
DHFR Dihydrofolate reductase (DHFR), this 25kDa protein is involved in the thymidine biosynthetic pathway. The purification of proteins with this tag can be achieved by methotrexate-linked resin.
mCitrine Although the earliest variant EYFP is still widely used, its high pKa value and sensitivity to halides make the application of EYFP unsatisfactory. Monomer variants of lemon yellow (mCitrine) and Venus (mVenus) are currently the most widely used yellow fluorescent proteins.
mRuby2 The brightest red fluorescent protein.
Neo The general kanamycin resistance gene encodes neomycin phosphotransferase II, the gene is denoted as neo, which can degrade G418, neomycin and kanamycin to achieve the corresponding resistance of eukaryotic and prokaryotic cells. Enzymes are special in genetic engineering. They can be expressed in prokaryotes to make Escherichia coli and others exhibit kanamycin resistance, and they can also be expressed in eukaryotes to make animal and plant cells exhibit G418 resistance.
Puro Puromycin (PM) is a protein synthesis inhibitor. It has a structure similar to the end of the tRNA molecule and can bind to amino acids instead of aminoacylated tRNA. It binds to the A site of the ribosome and is incorporated into Growing in the peptide chain. Although puromycin can bind to the A site, it cannot participate in any subsequent reactions, leading to the termination of protein synthesis and the release of an immature polypeptide containing puromycin at the C-terminal, which acts to inhibit the prolongation of protein synthesis.
B42,GAL4LexAVP16 These tags are used for protein interactions in the yeast two-hybrid system and can be used as DNA binding (GAL4, LexA) domains; transcription activators (B42, GAL4, VP16)