An epitope is a structural feature of a molecule that is specifically recognizable by an antibody and to which the variable region of the antibody binds. Short amino acid sequences, branched-chain carbohydrate groups, or even the phosphorylation site on a peptide can serve as an epitope. Most large molecules like proteins contain several epitopes and, when such an antigenic molecule is injected into an animal, it will induce the production of several different antibodies to many or all of these epitopes in that individual animal. Serum isolated from this individual will contain a mixture of several secreted IgG antibody proteins each produced by a different line of antibody-producing B lymphocytes and each capable of recognizing a different epitope of the antigen. This type of antibody is referred to as a polyclonal antibody because several different clones of antigen-producing B lymphocytes produce the several serum antibodies.
The advent of monoclonal antibody technology solved the problems of antibody heterogeneity and made possible the method of epitope tagging for protein detection. Monoclonal antibodies are produced by mouse lymphocytes. An antigen having multiple epitopes is injected into the mouse and B lymphocyte differentiation is allowed to initiate. Multiple B lymphocyte clones each producing a different antibody specific to a particular epitope of the antigen will begin to undergo this differentiation process. The immunized mouse is sacrificed, the B-lymphocyte-containing spleen removed, and the developing B lymphocytes fused with an 'immortal' line of B lymphocyte tumor cells. Normally, B lymphocytes are able to undergo only a very limited number of divisions in culture, but these tumor cell fusions divide indefinitely. Individual hybrid cell clones are cultured separately and allowed to produce their unique antibody. This immortalized antibody-producing B lymphocyte cell line is called a hybridoma. The antibody product of each hybridoma line is then tested against the antigen, and fragments of the antigen, and the specific epitope recognized by the antibody determined. The antibody product is referred to as a monoclonal antibody because it is produced by a single hybridoma clone of B lymphocytes.
A variety of epitope-specific monoclonal antibodies are commercially available but not all are equally useful for different protein analysis techniques. Some are excellent as the primary antibody in Western analysis while others are poor for this application but work extremely well in immunoprecipitation. One should be guided by the methods used by published journal articles and by the recommendations of colleagues and commercial suppliers. Commercial suppliers provide technical assistance for customers and detailed information can be found in the product descriptions of most catalogues. Since all monoclonal antibodies are murine antibodies, all contain the murine constant region. For techniques such as Western analysis that often use a monoclonal antibody as the primary antibody, the secondary antibody must be produced in an animal other than the mouse and directed against the mouse IgG constant region.
The most common type of epitope used by molecular biologists is the peptide epitope. This is because one can easily attach such an epitope to any protein of interest using recombinant DNA technology. This is referred to as epitope tagging. For this one simply needs to construct an in-frame fusion of the ORF of the gene encoding the protein of interest to an oligonucleotide encoding the epitope sequence. Most often, the epitope is placed at the N-terminal or C-terminal end of the ORF. It is essential to test the epitope-tagged allele for function to ensure that the presence of the epitope does not interfere with the functional activity, subcellular localization, or stability of the protein. In Saccharomyces this is done by determining whether the tagged allele is capable of complementing all of the mutant phenotypes of the null allele.
Vectors specific for constructing these in-frame epitope fusions can be obtained commercially or from colleagues. Many are described in the literature. These typically contain the epitope sequence located immediately upstream or downstream of a multiple cloning site (MCS). The ORF of interest is simply amplified using appropriate PCR primers and inserted into the MCS thereby placing the epitope at the N-terminus or C-terminus, respectively, of the encoded fusion protein. The resulting protein is said to be epitope tagged. Expression of the epitope-tagged gene product in these constructions is usually from a promoter such as the ADH1 promoter.
One need not use these vectors, particularly if one wants to use the native promoter. Because the epitope sequence is frequently quite short, one can synthesize oligonucleotide primers containing the sequence and use these for PCR amplification of the ORF of interest. This product can then be inserted into any vector containing any desired promoter sequence (see the discussion of expression vectors in Chapter 1). The sequence of the epitope also may be inserted into any desired site in the ORF of a gene using in vitro mutagenesis techniques or by PCR-based methods. This is important if the N-terminal or C-terminal versions of the epitope-tagged protein are not functional and another insertion site for the epitope must be found.
Longtine et al. (1998) constructed a series of modules for use as PCR templates for the creation of tagged fusion genes at genomic sites by one-step gene replacement. The modules allow for C-terminal fusion of GFP, three copies of the HA epitope, 13 copies of the Myc epitope, or GST or for N-terminal fusion of GFP, three copies of the HA epitope, or GST to the gene of interest. The N-terminal fusions replace the native promoter with that of GAL1. Each module contains the protein tag and a selectable marker. This is amplified using primers containing 40 bp of genomic sequence at the 5' end and sequence complementary to the module template and the 3' end. The amplified product is then transformed into the host strain and in vivo recombination between the amplified DNA fragment and the genomic site creates the desired tagged fusion gene.
The researcher has a choice of any of several peptide epitopes. The most common are listed below. Monoclonal antibodies specific to each is commercially available. For some, the researcher has a choice of different monoclonal antibodies based on their performance in a particular technique such as Western analysis or immuno-precipitation or even whether the epitope is at the N-terminal or C-terminal end of the protein or in the middle.
HEMAGGLUTININ (HA) EPITOPE
The HA epitope is a nonapeptide (YPYDVPDYA) derived from the influenza virus hemagglutinin protein. Anti-HA antibody is quite specific and cross reaction to other yeast proteins is not seen. The HA-tag can be used for Western analysis, immunocytochemistry, and immunoprecipitation (see below). Often, the HA epitope sequence is repeated up to three times or more to improve antibody binding and make this tag more useful for immunoprecipitation.
The FLAG epitope (DYKDDDDK) is recognized by three commercially available monoclonal antibodies, Ml, M2, and M5. Ml and M5 require that the epitope be placed at the N-terminus of the tagged protein and Ml will even bind to a shorter version of the FLAG sequence. M2 is able to recognize the epitope at all locations in the protein. Moreover, M2 can be used for both Western analysis and immunoprecipitation.
The Myc epitope (EQKLISEEDL) is derived from the human Myc protein, the product of the myc oncogene. A number of different monoclonal antibodies are available from commercial sources. Some are suitable for Western analysis and immunoprecipitation while others are more suitable for immunocytochemistry.
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