April 1, 2007 (Vol. 27, No. 7)

Now an Essential Component of Biotech Research, It Could Lead to New Therapies

Epitope tagging technology occupies a pivotal position in the biotechnologist’s repertoire. It is a reporter system in which an easily recognizable peptide sequence is affixed to a target, allowing it to be followed with precision. It can be used to localize proteins within the cell, identify interactions with other proteins, determine the function of a protein, and predict its fate. Epitope tags excel in protein purification using affinity columns. Since epitopes are well described and thoroughly studied, a wide range of commercial antibodies that bind to them are available.

Strategies for Disease Treatment

Interest in epitope tagging technology is rife since there is widespread recognition that crucial cellular processes are accomplished by large protein complexes within the cell. These dynamic molecular machines are constantly breaking up into components and reassembling in order to form massive collective protein groups that control critical functions within the cell.

According to Ira Chang, Ph.D., at the University of Nevada, Reno, “We can genetically engineer the proteins of model organisms, such as Arabidopsis, with epitope tags. By using antibody affinity columns and mass spectrometry, we can determine vital events pivotal for malignancy and diseases of aging, such as Alzheimer’s.”

According to Dr. Chang, 90% of the genes in Arabidopsis code for proteins of unknown function and represent a potential wealth of biotech products. However, the dissection of the “interactome” is a vital step in moving forward with this understanding, so the use of epitope tags is an essential tool for isolating and identifying the attendant proteins.

The approach employed by Dr. Chang and his colleagues involves transfecting ribosomal genes with epitope tags into the Arabidopsis strains. These powerful molecular magnets can be pulled out from cellular mixtures using appropriate anti-tag antibodies that allow one-step purification. Mass spec analysis then identifies the proteins that are complexed with the original tagged protein. Among suppliers of epitope tagging procedures, Sigma-Aldrich (sigmaaldrich.com) markets a mass spec-compatible kit.

“Epitope tagging combined with mass spec analysis is a powerful tool for the systems biology approach to understanding complex diseases,” Dr. Chang states.

Handle with Care

Epitope tags have been exploited by NeoClone (www.neoclone.com) in an innovative immunoaffinity-chromatography-purification protocol. Frequently, binding of the epitope tag to its corresponding antibody is so tight that it is extremely difficult to elute it from the bound Mab. The harsh elution conditions will frequently damage or disrupt protein structure, resulting in low yields of fully active, purified protein. Not only does this decrease the chance of purifying the protein in its native, functioning state, but also, these procedures wreak havoc on the antibody column, drastically lowering its useful lifespan.

To meet this challenge, NeoClone scientists have engineered a special class of polyol-responsive Mabs, which have the ability to gently offer up the protein-of-interest to which they are bound so it retains its full biological activity along with other accessory proteins with which it is functionally associated. The company has designed a corresponding class of epitope tags, Softag™, which bind to these antibodies and are now available. Developed by Richard Burgess, Ph.D., of the University of Wisconsin, they are now licensed to NeoClone.

“The Softag system allows a gentle release adaptable to a wide range of recombinant protein purification protocols,” states Mike Zwick, Ph.D., vp of business development at Neoclone. “We have designed a panel of Eukaryotic tags for use in prokaryotic protein isolation and vice versa.”

Most of these are offered by NeoClone as antibody resins for protein purification or protein-protein interaction studies. NeoClone also has a rapid process for the development of custom monoclonal antibodies and specializes in selection of polyol antibodies, which are especially useful for immunoaffinity purification.

The company forged a partnership with Pierce Biotechnology(www.piercenet.com), a unit of Thermo Fisher Scientific (www.thermofisher.com), to develop a series of antibodies for the purification and detection of fusion-tag proteins. According to their agreement, NeoClone will build the antibodies and Pierce will be responsible for a series of kits based on the NeoClone reagents. Neoclone also develops Mabs on a fee-for-service basis by using a retrovirus transformation protocol.

Better Affinity Purification

Kerstin Crusius, Ph.D., heads a research team at Bayer Schering Pharma (www.bayer.com) involved in development of novel epitope tags. “Although protein tags, such as GST and maltose binding protein, are commonly used as fusion partners for the expressed protein, removing the protein tag often results in precipitation of the protein of interest,” Dr. Crusius reports.

For this reason, the Crusius group has investigated the use of short peptide sequences that usually do not impact protein solubility, so the proteins remain in solution even after tag removal. Although many of the available short tags, such as Flag and c-myc, are reliable, the application of the antibodies, as well as the column matrices, to large-scale projects can be extremely pricey.

To meet this challenge, the team investigated various peptide sequences, focusing on a seven amino acid polypeptide tag that corresponds to the NH2 terminal sequence of human TGF alpha. Referred to as Tab2, it was used as a target for developing Mabs. The tag-antibody combination proved to be quite effective in immunoaffinity protocols and a variety of tasks, including Western blotting, FACS analysis, and immunoprecipitation.

However, the anti-Tab antibody binds extremely tightly to Tab, which made elution from affinity columns difficult, requiring high pH conditions. For this reason, Dr. Crusius designed an even smaller tag, referred to as Tab2s, consisting of only five amino acids. The Tab2s peptide binds the same Mab, but can be eluted under milder conditions, and in addition, does not affect the biological activity of the target protein to which it is fused.

The Tab2 and Tab2s tags are comparable to other tags in many protein detection and purification methods, but offer considerable economic advantage for use over a broad spectrum of applications, according to Dr. Crusius. The antibodies are available to qualified investigators through a material transfer agreement.

Manipulation of Determinants

The design and alteration of epitope tags and the genes they highlight has been streamlined and expedited through an array of commercial technologies. According to a number of scientists interviewed for this article, the Gateway™ technology (invitrogen.com/gateway) from Invitrogen (www.invitrogen.com) has facilitated the manipulation of epitope tags. Gateway is a universal cloning technology that allows the transfer of DNA segments through a variety of cloning vectors with simplicity and high efficiency. This feat is accomplished through use of the attP integration site in Phage Lambda and its partner bacterial integration site attB in E. coli.

The gene of interest is first cloned into an archival vector (Entry vector), which contains attL recombination sites just upstream and downstream of the inserted gene. From here, the gene fragment, or ORF, can be moved into any vector containing attR site (Destination vector). Destination vectors typically contain upstream promoters required to initiate transcription in specific hosts and epitope tags either at the 5´ or 3´ ends of the gene.

The protocol is fast and simple; to subclone the gene of interest from the entry clone to the destination vectors, the two are mixed together and incubated in the presence of the cloning enzyme mixture for one hour. The company supplies a range of destination vectors with various epitope tags for use in in vitro, cell-free E. coli, yeast, insect, mammalian, adeno, and lentiviral systems.

Hundreds of other Destination vectors have been developed by academic laboratories across the world. “Currently, Invitrogen is the only commercial provider of Gateway Destination vectors,” reports Balwant Patel, Ph.D., business area manager for cloning and expression products and services at Invitrogen.

Invitrogen recently extended a license to Covalys Biosciences (www.covalys.com) for the commercialization of Destination vectors containing the SNAP-tag technology for specific protein labeling in living cells. According to Dr. Patel, Invitrogen is in the process of liberalizing its licensing policy and is open to discussions with other life-science providers who may be interested in supplying their own Gateway Destination vectors to researchers in academia and industry.

The technology takes advantage of two types of expression vectors, supporting either the expression of untagged proteins or fusion proteins. For the various vectors, different expression elements may need to be installed between the attB sites prior to the Gateway reactions.

The basic Gateway technology, available since 1999, has been improved to allow for simultaneous cloning of multiple DNA fragments. The Multisite Gateway Kit and Pro technologies enable the assembly of as many as four elements in a single plasmid in correct order and orientation. This is accomplished by increasing the number of available att recombinational sites. These modifications of the original Gateway technology make it quite useful for the expression of proteins with different fusion partners and epitope tags.

Unique Protein Identification

“Lack of appropriate content is a major bottleneck in protein identification,” according to Neal Gordon, Ph.D., president of Epitome Biosystems(www.epitomebiosystems.com). “Most of the probes that are used for measuring proteins in multiplex modes are invariably antibodies, and these antibodies may lack the necessary specificity for adequate performance.”

Ordinarily, antibodies for immunoassay platforms are generated by immunization of animals with the entire protein, so many different and unknown epitopes participate in the process. These epitopes may be shared with many proteins and lack the requisite specificity.

Epitome’s technology involves identifying unique peptide sequences, or EpiTags™, in the target protein, generating these peptides, and using them in the immunization process. The company uses an informatics approach to identify the EpiTags from sequence information, so each is chemically synthesized and then used as an immunogen to generate highly specific anti-peptide antibodies. Since most proteins have multiple sites that will serve as EpiTags, multiple antibodies may be generated to each protein.

“Essentially, by removing or liberating the targeted structures from the protein, we turn the problem on its head,” Dr. Gordon continues. “We generate discreet peptides that represent ideal targets for immunization. This strategy allows us to create antibodies that work well in multiplex assays where we may target 10 to 15 proteins simultaneously.”

The specificity of the approach is greatly enhanced through the use of the sandwich assay, in which an antibody is immobilized on a solid surface to capture the protein, and a second antibody is used as a detector. The Epitome platform effectively addresses the need for a broad profile, a requirement for high levels of quantification, and the need for high levels of specificity. This is especially critical in the identification of phosphorylated proteins, which represents a focus area for Epitome. The phosphorylation status of proteins is important in the evaluation of cancer therapeutic agents.

The EpiTag antibodies can be configured into multiplex assay systems. A standardized platform for all proteins is employed that includes enzymatic digestion of the sample in order to liberate EpiTags for efficient binding to the antibody. The result is an assay procedure that measures and identifies peptides rather than proteins, avoiding many of the problems of low specificity that result in confusing and erroneous data.

Epitope Tags Rising

In the three decades since the concept of epitope tags was described in the scientific literature it has grown into an essential component of biotech research. Through the use of immunofluorescence, tagged proteins can be localized in cells and their movement through the cell analyzed.

Protein-protein interactions can be determined through the formation of multiprotein complexes with tagged proteins. The function of tagged proteins can be determined through activity assays. This wide array of tools for new product development continues to grow and is one of the most promising approaches for developing innovative therapeutic modalities. The speed, flexibility, and economy of peptide epitope tags make it possible to gather information about proteins that could only be assembled with great effort using conventional approaches.

K. John Morrow, Jr., Ph.D., is president of Newport Biotech.
E-mail: [email protected]

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