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How to Select Protein Tags

Selection of an ideal protein tag or reporters can be essential for the validation of a new experimental AAVs, or even an essential component of experimental design. PackGene offers a library of proteins tags and reporters for integration into your custom AAV, including those listed with potentially useful information in the table below.
Tag Names Introduction to protein tags
eGFP/eCFP
/eYFP/mCherry
Commonly used fluorescent protein tags include enhanced green fluorescent protein (eGFP), enhanced cyan fluorescent protein (eCFP), enhanced yellow-green fluorescent protein (eYFP), and monomer red fluorescent protein (mCherry). These fluorescent proteins have been engineered from wild-type fluorescent proteins and differ across their excitation wavelengths and emission wavelengths. The fluorescent protein eGFP is commonly selected as a protein fusion tag or as a reporter protein due to its high florescence intensity and long-established history as a relatively inert when expressed in cells. Nevertheless, it has been shown that eGFP may occasionally interfere with normal protein function when used as a fusion protein tag. Alternatively, mCherry is a relatively small red fluorescent monomer, and several studies have demonstrated negligible disruptions in protein function when mCherry is fused with foreign proteins at the N-terminus and C-terminus.
tdTomato Tandem dimeric tomato (tdTomato) is often considered the brightest and most stable amongst commonly used fluorescent protein tags. tdTomato consists of two copies of the fluorescent dimer dTomato connected by a 12 amino acid linker sequence. While tdTomato holds high brightness and fluorescent stability, it is also quite large relative to other protein tag options. The overall size of tdTomato may limit its usefulness for some applications since fusion of the large protein may interfere with folding and function of the target protein.
mCitrine
mVenus
Both mCitrine and mVenus are monomer variants of the yellow fluorescent protein lemon yellow. These variants are much smaller than the commonly used lemon yellow variant EYFP, and therefore may be more suitable as protein tags in some cases.
Flag Flag fusion tag is a short peptide composed of eight amino acids (DYKDDDDK). The Flag tag was specially designed for immunoadsorption purification of recombinant proteins, and thus was engineered as a short structure containing an enterokinase cleavage that lies between the Flag and the fusion protein. With this design, the Flag Tag can readily be removed from the fusion protein following purification by incubation with enterokinase.
aHis6 6xHis tag, also known as polyhistidine, His6 or hexahistidine tag, consists of 6 tandem Histidine residues and can be placed on the C- or N-terminus of a target protein. This relatively small tag holds low immunogenicity, low hydrophobicity, and low variability in tertiary structure when exposed to a wide range of detergents or other additives. Due to these features, recognition of a 6xHis can be achieved under both native or denatured conditions. Target proteins fused with 6cHis tags will bind to fixed ions such as nickel, cobalt, or copper when incubated under specific buffer conditions. Further manipulation of buffer conditions prompts the release of both the 6xDis tag and the fused target protein. Thus, an 6xHis tag provides a mechanism for separation and purification of 6xHis-labeled target proteins without the need for protein-specific antibodies or probes.
c-Myc C-Myc, or simply Myc tag is a small tag (EQKLISEEDL) that can be fused to the C- or N-Terminus of a target protein. Several high-quality commercial antibodies are available for detection of C-Myc by immunochemical methods.
HSV Herpes simplex virus (HSV) is derived from glycoprotein D precursor envelope protein. This relatively short tag (QPELAPEDPED) can be fused to the N- or C-Terminus and is unlikely to interfere with target protein structure or function. Several commercial primary antibodies are available for detection of HSV by immunochemical methods.
MBP Maltose binding protein (MBP) is a relatively large protein tag (43kDa) that substantially increases target protein solubility, and is one of the most common fusion protein tags used in microbes. MBP forms a strong bond with low-cost amylose resins, and can be eluted by the addition of maltose to achieve target protein purification.
T7 This is a 260-residue tag from the gene 10 fast service of T7 phage; it can enhance the expression level in E. coli.
SV40 NLS SV40 NLS is a nuclear localization signal peptide (PKKKRKV or PKKKRKVG) that was originally derived from simian virus 40 large tumor antigen (SV40 large T antigen). Fusion with SV40 NLS will enhance nuclear entry of the target protein.
P2A/T2A P2A and T2A are 18-22 amino acid long self-cleaving peptides. While the precise mechanism of P2A/T2A self-cleavage remains unconfirmed, the net effect of incorporating these elements is functional protein cleavage at the P2A/T2A site. Incorporation of P2A/T2A thus allows for a single transcribed mRNA to generate two separate proteins. Incorporation of P2A/T2A elements is particularly useful for the study of proteins that lose native function when fused with protein tags. Specifically, the incorporation of P2A/T2A between the target protein and a reporter protein allows for simultaneous transcription and translation of two proteins without compromising the function of the target protein.
IRES The internal ribosome entry site (IRES) is an RNA structure that is capable of independently recruiting ribosomes to drive translation of downstream mRNA. Thus, the incorporation of an IRES element provides a mechanism for a single transcribed mRNA to generate multiple separate proteins. More specifically, IRES elements within an mRNA function as independent transcription initiation sites such that one protein may be transcribed by traditional Cap based mechanisms while addition proteins may be transcribed from that same mRNA via the IRES dependent mechanism. Incorporation of IRES elements thus provides a mechanism for simultaneous transcription of multiple proteins with independent translation of each.
V5 The V5 tag is a 14 amnio acid long peptide tag (GKPIPNPLLGLDST) derived from P/V protein of Paramyxovirus SV5. The V5 tag can be fused and expressed on the N-terminus or C-terminus of a target protein for analysis and identification. Several commercial antibodies are available for detection of V5 by immunochemical methods and conjugated beads are commercially available for immunoprecipitation via V5.
CAT Chloramphenicol acetyltransferase (CAT) is a 24kDa enzyme responsible for bacterial resistance to the antibiotic chloramphenicol. The enzymatic function of CAT can be used to measure absolute expression of the CAT enzyme by monitoring acetylation of radioactively labeled chloramphenicol. CAT is most often used as a reporter, but also retains its enzymatic function when used as a protein fusion tag, and thus fusion with a CAT protein tag can be used to directly measure target protein expression levels without the need for PAGE or an immunoassay.
DHFR Dihydrofolate reductase (DHFR) is a 25kDa protein is involved in the thymidine biosynthetic pathway. The purification of proteins with this tag can be achieved by methotrexate-linked resin.
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)