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Jul 1, 2006 (Vol. 26, No. 13)

Epitope Tagging Generates New Products

Tracing the Flow of Biological Molecules through Living Systems

  • Epitope tags are proving to be an important tool for tracing the flow of biological molecules through living systems. An epitope (or antigenic determinant) is a biological structure or sequence, such as a protein or carbohydrate, recognized by an antibody as immunogenic. With the aid of appropriate bacterial strains and vectors designed for the purpose, a fusion protein can be generated consisting of the protein of interest bonded to a small peptide fragment comprising a new epitope. This peptide is one of a stable of choices selected for its antigenicity, and proteins tagged with it can then be detected using a specific commercial antibody produced for this purpose.

    Epitope tagging offers a number of advantages over conventional strategies for identifying and tracking gene products. The prep time is much shorter than that encountered during conventional antibody development, since it is unnecessary to produce a new antibody every time a different protein is studied. Using the epitope tag, a variety of proteins can be detected by the same tag-specific antibody.

    The costs of tagging are considerably less, and epitope tagging can be used to distinguish between two proteins with similar antigenicity. There are numerous protein families, such as the hemoglobins, whose members are homologous and difficult to distinguish immunologically. The technique provides a purification method for rare and difficult to isolate proteins that allows one-step purification.

  • Epitope Tags for Protein Purification

    For the biotechnology industry, epitope tags are a formidable tool in protein purification. A fusion protein, composed of a protein of interest and a well-defined tag, can be overexpressed in a bacterial or mammalian cell and rapidly purified using affinity resins. In some cases the smaller tags do not interfere with protein performance and do not have to be removed, whereas larger tags will increase protein solubility but may have to be cleaved off, adding an additional step to the protocol.

    "In recognition of the demand for flexible and integrated epitope-tagging systems, Sigma-Aldrich has established a portfolio including the HIS-Select and FLAG epitope-tagging technologies," states Dr. Richard Pembrey, Ph.D., market segment manager at Sigma-Aldrich (www.sigmaaldrich.com).

    HIS-Select is a patented technology based on a noncharged hydrophilic linkage chemistry that provides greater binding capacity and reduces nonspecific binding commonly associated with charged chelate linkages. Sigma addresses the various research needs by providing capture and purification systems for histidine-tagged proteins. They also cater to pharmaceutical companies by offering custom packaging and batch validation.

    "In addition to our range of various epitope tags, including GST, c-Myc, and V5, we upgrade our products to meet research needs," Dr. Pembrey continues. "Recently, we expanded our FLAG epitope product line by introducing the new affinity purified FLAG M2 antibody, addressing the needs of proteomic researchers by providing greater sensitivity for advancing discovery."

    The HIS-Select line is based on a Sigma noncharged hydrophilic linkage chemistry with higher binding capacity that reduces nonspecific binding commonly associated with charged chelate linkage. Sigma-Aldrich also carries an extensive line of anti-epitope Mabs.

  • Focused Phosphatase Tagging

    Strategene (www.stratagene.com) recently introduced a product aimed at elucidating the role of phosphatases in cellular regulation, according to Carsten Carstens, Ph.D., scientific program director. "We brought to market our Signal Scout Phosphatase Reagents in late 2005. These are a series of expression vectors using the myc tag for tracking the role of phosphorylation in cellular regulation."

    Phosphatases remove phosphate groups from protein, profoundly modifying their activity. These changes have significant effects on a vast assortment of cellular processes, including malignant transformation, cellular senescence, and inflammation. The Signal Scout system is a series of vectors carrying a wide selection of fusion phosphatases with myc tags, mutated to bind their target phosphate group. When introduced into the cell, they trap the phosphorylated protein, yielding measures of cellular regulatory processes.

    "With Signal Scout, the investigator can determine whether phosphorylation is involved in a particular cellular activity," says Dr. Carstens. "In our Signal Scout Phosphatase Profiling Systems I and II, we have created an all-in-one-kit to facilitate phosphatase research."

    Each kit includes a c-myc-tagged human phosphatase expression clone, a c-myc-tagged substrate trapping mutant clone, and an anti-c-myc antibody.

    "High performance anti-tag antibodies are essential for the detection, characterization, and purification of proteins," according to George McBride, director, biotools business segment for Chemicon International and Upstate (Serologicals;www.serologicals.com). "Many companies produce them in conjugated forms that recognize widely exploited sequences such as human c-myc protein, glutathione-S-transferase, six histidine residues (His6) influenza hemagglutinin protein, and calmodulin binding protein. These sequences may be inserted at the desired position within the protein-coding sequence by conventional molecular DNA technologies."

    Chemicon and Upstate have developed a number of high-affinity antibodies for the detection and purification of many epitope tagged proteins. According to the companies, its anti-c-myc antibody (clone 4A6) has proven quite successful in manipulating these compounds.

  • c-myc

    The human c-myc gene has been favored over many other tags most likely for historical reasons dating back to the 1982 discovery of c-myc in library screening. In 1985 the continuing identification of c-myc as an important oncogene stimulated a vigorous program to generate higher quality antibodies for its identification, assay development, and the exploration of its role in oncogenesis. Hence, antibodies to the fusion-tags were readily available to monitor fusion protein expression, co-localization, and purification.

    Among these, clones 4A6 and 9E10 have proven of particular value. They have been used in western blotting, immunoprecipitation, and flow cytometry. The 4A6 antibody, raised to a smaller consensus myc tag sequence, recognizes myc tag on either the amino- or carboxyl-terminus of a recombinant protein. Its ability to recognize myc tag is independent of the sequence following or preceding the tag.

    The 4A6 clone has been successfully employed in immunofluorescence protocols. C-myc-tagged proteins can be affinity purified by coupling the Mab 4A6 cross-linked to protein G agarose by dimethyl-pimelidate. So over the years myc has acquired a role as one of the universal tags much like secondary antibodies.

    Clone 9E10 has long been valued as a tool for the detection of the myc-epitope tag during analysis, expression, and purification of recombinant proteins. However, its ability to recognize N-terminally myc-tagged proteins is dependent on the amino acid sequence following the myc-epitope tag sequence. This may be because the 9E10 antibody was raised against a larger peptide from human c-myc (residues 408-432), and therefore not as specific to the primary myc sequence.

    Upstate offers an exclusively licensed anti-myc tag, clone 4A6 monoclonal antibody, which provides better results than the monoclonal 9E10. Clone 4A6 detects myc-tagged recombinant protein in sequence contexts not well recognized by anti-myc tag, clone 9E10. These reagents offer investigators a range of experimental possibilities.

    EMD Biosciences (www.emdbiosciences.com) handles a range of epitope-tagging products. Among these is the His•Tag Antibody Plate, a 96-well, ELISA-compatible plate containing an immobilized His tag monoclonal antibody, covalently immobilized to the surface to retain maximal binding activity. The antibody specifically recognizes five consecutive histidines, and so will bind with high affinity to virtually any His•Tag fusion protein in which the tag is exposed, according to the company. Well-to-well variability is less than 5% and the plates are stable when stored dry at 4C. The His•Tag Antibody Plate can be used in a variety of binding assays where immobilization of His•Tag fusion proteins is required.

  • Cleaving Tags from Fusion Proteins

    One of the major problems facing users of tagged or fusion proteins is the removal of the tag following protein purification. Tags can be most easily removed with specific proteases, when cleavage sites are built into the expressed protein between the epitope tag and the protein, but this process is often inefficient and may result in unwelcome degradation of the protein, if nonspecific protease activity is present. Balwant Patel, Ph.D., business area manager, gateway, TOPO, and protein expression of Invitrogen (www.invitrogen.com), explains how his company negotiated this challenge. "We use the SUMO protease, which recognizes the tertiary structure of the sumo protein rather than an amino acid sequence. The enzyme is highly active and very specific. It cleaves off the entire Sumo fusion protein sequence leaving the target protein with a native N-terminal end."

    SUMO is a small protein moiety discovered a decade ago that engages in a novel host cell post-translational modification system termed sumoylation, involving the attachment of SUMO to substrate proteins. Its unique removal by SUMO protease makes it an ideal epitope tag.

    The AcTEV System is an alternative designed for removal of solubility, secretion, detection, and purification tags from recombinant proteins. It is a modified version of the original Tobacco Etch Virus protease (TEV) that is significantly more stable than native TEV protease, but maintains high specificity and high activity. AcTEV Protease recognizes the seven amino acid sequence (E-N-L-Y-F-Q-G) and it cleaves between the glutamine and glycine. A six-histidine tag on AcTEV helps its removal after digestion of the target protein.

    Invitrogen also carries an extensive collection of expression vectors with epitope tags for construction of fusion proteins and accessory detection and purification antibodies. Many of the vectors are supplied in the Gateway recombination cloning format, which allows simplified transfer of DNA sequences into destination vectors for the construction of fusion proteins for expression in many different hosts (E. coli, yeast, mammalian cells, etc).

  • New Tools for Protein Analysis

    "Ciphergen (www.ciphergen.com) is committed to the development of new tools to aid investigators in the understanding of receptor structure and function," states Lee Lomas, Ph.D., director of biology research and development. "Although Ciphergen does not directly participate in the development of epitope tags, we do provide tools and reagents that allow these tags to be utilized for capture and detection directly by mass spectrometry."

    For example, two array chemistries provide the capability to covalently attach a ’bait’ molecule to Ciphergen’s ProteinChip arrays through a primary amine coupling mechanism. This allows antibodies specific to tags (e.g., anti-mycmic-IgG for mycmic tags), small molecules (e.g., glutathione for GST fusion tags) and avidin (for biotinylation tags) to be immobilized followed by the selective extraction of the tagged protein.

    Other types of interaction experiments include epitope mapping, whereby after capture of the protein of interest to the bait antibody, the complex is selectively digested, then washed to reveal the specific epitope sequence of the interaction and selective point mutation to demonstrate relative influence of specific amino acid residues within a binding site. This technology can be applied to modulation of off-site toxicity of peptide or protein therapeutics.

    "I also wish to stress our efforts in the Equalizer Bead Technology," continues Dr. Lomas. "The technique utilizes a combinatorial chemistry approach to generate affinity libraries of vast diversity. Each bead contains many copies of a single peptide ligand, used to selectively concentrate lower abundance proteins while removing the excess high abundant proteins. This approach increases the number of detectable proteins by threefold."

    Ciphergen has used this technology to identify several putative markers for ovarian cancer in serum samples from patients. These biomarkers form a panel currently under study in a large group of ovarian cancer patients.

  • Whither Epitope Tags?

    While the use of epitope tags is providing a powerful infusion of energy into the study of vital signaling proteins and leading to new ways of blocking disease-causing signals, it is important to add a cautionary note. The assumption is made that the tag is a neutral addition to the proteins under investigation and will not affect their movement, binding, or localization in the cell. This assumption, of course, lies at the heart of the epitope-tagging technology, since if the tags change the performance of the proteins under consideration, the investigator could be led to erroneous conclusions.

    However, studies using epitope tags on the human gonadotrophin-releasing hormone receptor have demonstrated that epitope tagging alters its cellular localization and can obscure the effects of other mutations on this vital protein. While it is widely held that mutations occurring in human patients cause the receptor to be misrouted to the plasma membrane, this effect is suppressed by epitope tagging.

    This observation has caused some molecular endocrinologists to accept a false conclusion, namely that mutations in the gonadotrophin receptor do not cause mis-direction of the receptor within the cell. Thus it is critical that these limitations of the technology be taken into account and controlled for. Otherwise companies in search of new drugs affecting cellular signaling may find themselves led down blind alleys.



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