How to Select Protein Tags
Selection of an ideal protein tag or reporter can be essential for the validation of a new experimental AAVs and may even be an essential component of overall experimental design. PackGene offers a library of proteins tags and reporters for integration into your custom AAV. Available protein tags are tabulated below along with potentially useful information.
|Introduction to protein tags
|Enhanced green fluorescent protein (eGFP), enhanced cyan fluorescent protein (eCFP), and enhanced yellow-green fluorescent protein (eYFP) are commonly used as both fusion-protein tags and as reporter proteins. These proteins have been engineered from wild-type fluorescent proteins and differ across their excitation 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, eGFP, eCFP, and eYFP are relatively large proteins when compared to alternative tag options. Importantly, the size these proteins may limit their usefulness for some applications, since fusion with such large tags may interfere with folding and function of the target protein.
|The dimeric red fluorescent protein tdTomato is one of the brightest and most stable fluorescent protein tags. tdTomato consists of two copies of the fluorescent dimer dTomato connected by a 12 amino acid linker sequence. While tdTomato is both bright and stable, it is also quite large relative to some other protein tag options. The size of tdTomato may limit its usefulness for some applications, since fusion with this large protein tag may interfere with folding and function of the target protein.
|The monomeric red fluorescent protein mCherry is a relatively small fluorescent protein derived from dsRed. Several studies have demonstrated negligible disruptions in target protein function when mCherry is used as a fusion protein tag on either the N- or C-terminus.
|The monomeric red fluorescent protein mRuby2 is a small fluorescent protein derived from eqFP611. This variant is smaller than other red fluorescent protein tag options and is also amongst the brightest available fluorescent protein tags.
|The monomeric yellow fluorescent proteins mCitrine and mVenus are monomer variants of the multimeric fluorescent protein lemon yellow. These variants are much smaller than the commonly used lemon yellow variant eYFP, and may therefore be better suited for use as a fusion protein tag.
|The light-emitting enzymes firefly luciferase (Fire-Luc), Renilla luciferase (Renilla-Luc), and Guassia luciferase (Guassia-Luc) are commonly used luminescent reporter proteins. The light emitted from these luminescent proteins reliably scales with protein expression levels, and thus luciferase reporters are often used for quantitative measurement of transcriptional activity. It is uncommon for these luminescent proteins to be used as fusion protein tags.
|The HA tag (YPYDVPDYA) is an epitope tag derived from human influenza virus hemagglutinin protein. The HA-tag is one of the most widely used epitope tags and can be fused to either the N- or C-Terminus of a target protein. The HA-tag forms a strong bond with commercially available antibodies for protein detection by immunochemical methods, and is commonly used to facilitate target protein purification via antibody-conjugated beads or gels. HA-tag fused proteins can then be eluted via high concentration of free HA peptide or by low pH buffer.
|The flag tag (DYKDDDDK) is an eight amino acid epitope tag that can be fused to either the C- or N-terminus of a target protein. The flag tag was specially designed for immunoadsorption purification of recombinant proteins, and was engineered to contain an enterokinase cleavage site that lies between Flag and it’s fusion protein partner. With this design, the flag tag can readily be removed from the target protein following purification by incubation with enterokinase.
|The 6xHis tag (HHHHHH), also known as polyhistidine, His6, or hexahistidine tag is an epitope tag that consists of 6 tandem Histidine residues. 6xHis can be fused to either 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 and denatured conditions. Target proteins fused with 6xHis 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 the 6xHis tag and elution of the target protein. Thus, the 6xHis tag provides a mechanism for separation and purification of 6xHis-tagged target proteins without the need for protein-specific antibodies or probes.
|The c-Myc tag (EQKLISEEDL) is a 10 amino acid long an epitope tag 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.
|The HSV tag (QPELAPEDPED) is an epitope tag derived from the glycoprotein D precursor envelope protein of herpes simplex virus. The HSV tag can be fused to the N- or C-Terminus of a target protein and is unlikely to interfere with target protein structure or function. Several commercial antibodies are available for detection of HSV by immunochemical methods.
|The V5 tag (GKPIPNPLLGLDST) is a 14 amnio acid long epitope tag derived from P/V protein of Paramyxovirus SV5. The V5 tag can be fused to the N-terminus or C-terminus of a target protein. Several commercial antibodies are available for detection of V5 by immunochemical methods and conjugated beads are commercially available for immunoprecipitation via V5.
|The T7 tag (MASMTGGQQMG) is an 11 amino acid long epitope tag derived from the leader sequence of T7 bacteriophage gene10 and is commonly used as a tag in expression vectors that use the highly efficient T7 RNA polymerase expression system. Several commercial antibodies are available for detection of T7 by immunochemical methods.
|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.
|Glutathione S-transferase (GST) is a 26kDA protein tag that facilitates target protein folding and inhibits target protein aggregation. GST forms a strong bond with its native substrate glutathione, and glutathione conjugated beads can be used to purify GST-tagged target proteins. Glutathione bound GTS-fusion protein can then be eluted with high concentrations of free glutathione.
|Maltose binding protein (MBP) is a 43kDa protein tag 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.
|Chloramphenicol acetyltransferase (CAT) is a 24kDa enzyme protein tag. The enzymatic function of CAT can be used to measure the absolute expression level of CAT by monitoring acetylation of radioactively labeled chloramphenicol. CAT is commonly used as a reporter, but also retains its enzymatic function when used as a protein fusion tag. Fusion with CAT protein tag can therefore be used to indirectly measure target protein expression levels without the need for PAGE or an immunoassay.
|Dihydrofolate reductase (DHFR) is a 25kDa protein tag. DHFR is an important enzyme in the thymidine biosynthetic pathway, and DHFR lacking cells cannot survive in medium without thymidine. DHFR lacking cells that have been successfully transfected with vectors that express DHFR as a reporter or target protein tag can thus be selected for by incubation with thymidine-lacking medium. This approach has proven particularly useful for applications involving DHFR lacking CHO cells. Purification of DHFR-tagged fusion proteins can then be achieved through methotrexate-linked resin.
|The Neo gene encodes neomycin phosphotransferase which is an enzyme that confers kanamycin, neomycin, and G418 resistance. Expression of Neo in prokaryotes provides a mechanism for expression selection via incubation with Kanamycin and Neomycin while eukaryotic cell expression selection can be achieved via incubation with G418.
|Puromycin is a protein synthesis inhibitor and antibiotic that is toxic to both prokaryotic and eukaryotic cells. Expression of puromycin N-acetyl-transferase via the Puro gene confers resistance to Puromycin, and thus provides a mechanism for expression selection in both prokaryotic and eukaryotic cells.
|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.
|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.
|These proteins are critical pieces for experiments that aim to use the yeast two-hybrid system and can be used as DNA binding (GAL4, LexA) domains or as transcription activators (B42, GAL4, VP16).