epitope tagging

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Annu. Rev. Genet. 1998. 32:601–18 c 1998 by Annual Reviews. All rights reserved Copyright °

EPITOPE TAGGING Annu. Rev. Genet. 1998.32:601-618. Downloaded from arjournals.annualreviews.org by University of Pittsburgh on 08/09/06. For personal use only.

Jonathan W. Jarvik and Cheryl A. Telmer Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213; e-mail: [email protected] KEY WORDS:

epitope tagging, protein fusion, gene fusion, antibodies, proteomics

ABSTRACT Epitope tagging is a recombinant DNA method by which a protein encoded by a cloned gene is made immunoreactive to a known antibody. This review discusses the major advantages and limitations of epitope tagging and describes a number of recent applications. Major areas of application include monitoring protein expression, localizing proteins at the cellular and subcellular levels, and protein purification, as well as the analysis of protein topology, dynamics and interactions. Recently the method has also found use in transgenic and gene therapy studies and in the emerging fields of functional genomics and proteomics.

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EPITOPES AND ANTIBODIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Locating the Tag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introducing the Tag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detecting the Tag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . APPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Topology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Genomics and Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Annu. Rev. Genet. 1998.32:601-618. Downloaded from arjournals.annualreviews.org by University of Pittsburgh on 08/09/06. For personal use only.

INTRODUCTION Epitope tagging, first described by Munro & Pelham in 1984 (68), is a recombinant DNA method for making a gene product immunoreactive to an already existing antibody. The process typically involves inserting a polynucleotide encoding a short continuous epitope into a gene of interest and expressing the gene in an appropriate host. Epitope tagging has become a standard molecular genetic method for enabling rapid and effective characterization, purification, and in vivo localization of the protein products of cloned genes. Epitope tagging has proved to be an efficient way to bring powerful immunochemical and immunocytochemical methods to bear on the protein encoded by a gene that has been cloned. The alternative and more traditional approach— raising antibodies to the encoded protein itself—is usually successful, but it also is slow, costly, and unpredictable. One first has to obtain sufficient immunogen, either by expressing the gene and purifying the encoded protein or chemically synthesizing a peptide representing a portion of the protein. These molecules are then used to raise polyclonal or monoclonal antibodies—processes that require many months and do not always readily yield reagent quality antibodies. Even when antibodies are isolated by more rapid methods such as phage display or in vitro immunization, one still must deal with the fact that every antibody is distinct with respect to such parameters as class, titer, affinity, avidity, background, response to temperature, pH, buffer conditions, etc, and so each new antibody must be individually characterized before experimental use. With epitope tagging, by contrast, one possesses the requisite antibody and knows its properties from the outset. Epitope tagging is not just a poor man’s way of doing immunochemistry and immunocytochemistry; there are also a number of circumstances in which use of traditional antibodies is inferior to epitope tagging or simply inappropriate. Not infrequently an antibody raised against a protein cross reacts with related or unrelated proteins, making analysis difficult or impossible. Epitope tagging overcomes this problem. In some cases, one wishes to distinguish the product of a transgene from the product of the endogenous gene. This distinction cannot be made using antibodies to the native protein but is readily achieved through epitope tagging (53, 81). In other cases, the protein of interest is a poor immunogen (90, 107) but can be made highly immunoreactive using epitope tagging. An epitope-tagged protein is a special kind of fusion protein in which the added amino acids are few in number (typically 6 to 30) and do not add new biological activity to the protein. These features make it particularly likely that a protein carrying the tag will retain, to a large degree, normal structure and function. But this is not to say that proteins with large additions cannot retain

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normal function and thereby provide many of the benefits of epitope-tagged proteins. Indeed, many fusion systems—involving, for example, fusion to β-galactosidase, alkaline phosphatase, glutathione S-transferase, Protein-A, and green fluorescent protein—exist in which the fusion partner is large, often larger than the target protein, and has a distinct biological activity of its own. Although these systems are not the subject of this review, it is important to point out that many fusion proteins made using these systems have shown localization and activity patterns characteristic of the target proteins themselves, making them, like epitope-tagged proteins, very informative molecules for examining protein function in vivo and in vitro.

EPITOPES AND ANTIBODIES Many validated epitope-antibody combinations are commercially available for use in epitope tagging, allowing considerable room for choice by the researcher (Table 1). This variety is good, because no epitope tag is ideal for all applications. For some epitopes, only one antibody is available; for others there are multiple choices, including monoclonals and polyclonals. Most of the available antibodies recognize the epitope whether it is internal to the protein or at a terminus, but in some cases recognition is dependent upon location, e.g. the tag is only recognized when at the extreme C terminus. We have not included information about this property for each antibody in Table 1 and advise the interested reader to consult the literature and/or the commercial suppliers. Some tags are not useful in certain applications owing to high background reactivity. Whether or not background immunoreactivity represents a significant problem in a new application is best addressed empirically by assessing the background reactivity in the conditions of interest prior to tagging. One of the tags listed in Table 1—the 15–amino acid S•Tag—is not an epitope tag in the strict sense but does have the essential features of an epitope tag— small size and specific recognition by a detecting molecule. This tag is derived from a portion of pancreatic ribonuclease A and is recognized not by an antibody but by a polypeptide representing a different portion of the ribonuclease protein. Another tag that is not strictly an epitope tag but is worthy of mention here is the 13–amino acid recognition site for in vivo biotinylation by Escherichia coli biotin holoenzyme synthetase. This tag is biotinylated in E. coli at a specific lysine residue, allowing the tagged protein to be detected and/or purified using avidin or streptavidin (105). Sensitivity is an important issue in epitope tagging. One way to increase sensitivity is to use tandem copies of the tag instead of just one. This significantly improves the signal strength and the signal-to-noise ratio. Tandem tags have been very popular, and numerous examples of successful tagging with

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Table 1 Commercially available epitope/antibody combinations Epitope name


AU1 AU5 BTag c-myc

Size

Sequence

6 aa 6 aa 6 aa 10 aa

DTYRYI TDFYLK QYPALT EQKLISEEDL

Antibody

Immunogen

IRS 5 aa KT3 6 aa Protein C 12 aa S•Tag® 15 aa T7

RYIRS PPEPET EDQVDPRLIDGK KETAAAKFERQH MDS 11 aa MASMTGGQQMG

BPV-1 BPV-1 VP7 of bluetongue viruses Synthetic peptide: residues 408-439 of human c-myc gene product M1, M2, M5 Synthetic peptide containing enterokinase cleavage site GLU-GLU Synthetic peptide EEEEYMPME of polyoma virus medium T antigen 12CA5 Human influenza virus 3F10 hemagglutinin, HA1 HA.11 fragment, aa75-aa110 6-His, 6xHis, Recombinant His-tagged HIS-11 fusion protein HSV•TAG® Synthetic peptide from HSV glycoprotein D PHHTT Synthetic peptide PHHTTPHHTTPHHTT IRS1 Synthetic peptide KT3 SV40 large T antigen HPC4 Human Protein C See text S-protein T7•Tag®

V5 VSV-G

14 aa GKPIPNPLLGLDST V5 6 aa MNRLGK P5D4

FLAG

8 aa DYKDDDDK

Glu-Glu

6 aa EYMPME or EFMPME

HA

9 aa YPYDVPDYA

His6

6 aa HHHHHH

HSV HTTPHH

11 aa QPELAPEDPED 6 aa HTTPHH

AU1 AU5 D11, F10 9E10

Leader peptide of phage T7 major capsid protein P/V of paramoxyvirus SV5 Synthetic peptide: C-terminal 15 residues of vesicular stomatitis virus glycoprotein

Reference 58 58 109 20

40 91

22

92

60 63 46 87

50

tandem double, triple, and even quadruple or quintuple tags exist in the literature (2, 18, 72, 99, 111). Does the use of multiple tags increase the risk that the insert will interfere with function? Intuition suggests that it will, but we are not aware of any published study that explicitly compares the effects of single epitope insertions to multiple ones. As stated above, there are many examples of well-tolerated multiple tag insertions. Perhaps larger tags are tolerated so well because in most cases they are in external loops or at termini where they do not greatly perturb the rest of the protein.

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Most epitopes that have been popular for epitope tagging are highly charged. For example, the 9-amino acid HA tag contains two aspartic acids; the 10amino acid c-myc tag contains three glutamic acids, one aspartic acid, and one lysine; and the 8-amino acid FLAG tag contains five aspartic acids and two lysines. Since one generally aims to place the tag in an external portion of the target protein, it is appropriate that the tag be charged rather than hydrophobic. However, a tag of extreme or inappropriate charge could cause problems in some cases, e.g. if a basic domain of a protein is tagged with an acidic sequence. Commercially available epitopes without highly charged amino acids include BTag, T7-Tag, and HTTPHH.

Locating the Tag MIDDLE OR ENDS? In the majority of cases described in the literature, the tag has been placed at, or very near, the extreme N or C terminus of the target protein. There are a number of reasons behind this choice of location. Some reasons are historical—the first proteins to be epitope tagged were tagged at the termini, and when new proteins were tagged it seemed wise to do what had worked in the past. Some of the reasons are practical—tagging is often performed using expression vectors that automatically put the tag at a terminus. And some reasons are theoretical—termini are frequently chosen in the belief that proteins will tolerate additions more readily at these locations than at other sites. While the latter belief may be true—termini are rarely included in active sites, for example—it is also true that many proteins are known for which terminal sequences are critical for function. The termini also appear favorable because they are likely to be on the outside of the folded polypeptide, where one wants the tag to go, and not in the hydrophobic core. But it must be remembered that, due to simple geometry, most of the amino acids in any protein are on the outside, and so, if a protein is tagged at a randomly chosen site, the tag will probably wind up on the outside anyway. It is clear that most proteins will accept a tag at one terminus or the other (or both) without severe loss of function. Based on our survey of the literature, we would like to provide the reader with a quantitative estimate of the likelihood that a terminal tagging event will be successful, i.e. will yield a functional, and properly localized, protein. Unfortunately, we cannot provide such an estimate because successful tagging events are more likely to be published than unsuccessful ones, and so the published literature is biased to an unknown degree. Interestingly, however, a survey of the literature does allow us to estimate the likelihood of success for internal tagging events. We can do this because a number of studies have been published in which peptides were added to proteins at randomly chosen internal sites and the effects on structure and/or function assessed. In these studies there was no bias in favor of reporting successes over failures.

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Table 2 Effects of internal guest peptides on protein function Protein

Insert size

HIV reverse transcriptase HIV reverse transcriptase E. coli met tRNA synthetase Adenovirus DNA polymerase Human acetylcholine receptor E. coli FhuA E. coli β-galactosidase Bovine rhodopsin E. coli LamB S. cerevisiae. Spa1 S. cerevisiae. Arp100 S. cerevisiae. Ser1 TEM-1 β-lactamase E. coli lactose permease E. coli lac repressor

3–9 aa 3–9 aa 5–14 aa 4 aa 8–45 aab 13 aab 27 aa 22 aab 12 aab 89–93 aab 89–93 aab 89–93 aab 5 aa 31 aab 31 aab

Tolerateda 10/23c 8/20d 4/8e 8/16c 11/13g 12/16h 3/16f 6/16i 8/9 j 12/13k 6/8k 9/13l 15/23m 10/21n 2/11o 124/196 (63%)

Not tolerateda 13/23c 12/20d 4/8e 8/16c 2/13g 4/16 h 13/16f 10/16i 1/9 j 1/13k 2/8k 4/13l 8/23m 11/21n 9/11o

References 85 85 100 9 1 67 3 6 75 89 89 89 30 62 74

72/196 (37%)

a

Criteria differ from case to case. In those cases where quantitative assays were performed, the tag is tabulated as tolerated if activity was greater than 30% of control. b Epitope tag. c Polymerase assay. d RNAse H assay. e Adenylate synthetase assay. f β -galactosidase assay. g Electrophysiological response to ACh. h Growth promotion by ferrichrome. i 11-cis-retinal binding. j Maltodextrin utilization. k Haploid cell viability. l Growth in serine-free medium. m Ampicillin resistance. n Lactose utilization. o Repressor activity.

Table 2 summarizes the results obtained in 16 such studies. In some cases the inserted sequence was an epitope tag; in others it was a peptide to which no antibody was available. The set of 16 is not comprehensive, but we believe it to be representative. Several conclusions can be drawn from the data in Table 2. First, every protein that was examined had some sites at which the insert did not dramatically disrupt function. Second, most proteins had many such sites; indeed, the aggregate data show that the tag was well tolerated in about 63% of all insertions. Third, and this was an explicit conclusion of several of the studies, multipass membrane proteins are especially tolerant of insertions in internal or external loops. Finally, there was no simple relationship between the size of the tag and

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how well it was tolerated; in many cases, a large tag was no more disruptive than a small tag. The 63% figure presented in Table 2 can be viewed as conservative. If one uses proper subcellular localization rather than activity as the criterion for success, the value is actually considerably greater than 63%. This is significant because in many, and perhaps most, applications of epitope tagging, the primary goal is to determine localization, not activity. Of course, if one is tagging a protein whose three-dimensional structure is known, the location of the tag can be chosen more rationally, e.g. the tag can be placed in an external loop or at a terminus removed from known active sites and from sites involved in known protein-protein interactions. For most proteins such information is not available, but even then some guidance can come from consideration of the protein’s primary sequence. Hydrophobic regions should be avoided as they are likely to be in transmembrane domains or in the hydrophobic core, neither of which is a good place for tagging. Likewise, highly conserved regions should be avoided because they are likely to be particularly sensitive to alterations in primary sequence.

Introducing the Tag The two standard approaches to tagging a cloned gene are: (a) An epitope encoding oligonucleotide is inserted into the coding sequence, or (b) the coding sequence is inserted into an expression vector that already carries the epitope tag. This review does not provide technical instruction with respect to cloning, but protocols can be found in many of the papers cited. When tagging by oligonucleotide insertion, it is important to take into account codon usage preferences for the target cell or organism. Expression vectors that incorporate epitope-tagging features have evolved from the early mammalian (80), yeast (23), and yeast/E. coli shuttle (86) vectors to a wide variety of specialized vectors developed by academic researchers (e.g. 24, 88) or developed in the private sector for commercial distribution. A survey of vectors available from the private sector (BAbCo, Boehringer Mannheim, Invitrogen, Novagen, Pharmacia, Promega, Sigma, Stratagene) finds expression vectors for mammalian, insect, yeast, and bacterial cells with a variety of epitope tags including c-myc, FLAG, HA, His6, Protein C, T7, V5, and VSV-G. In a number of vectors, the His6 tag is present in combination with another epitope, with His6 for purification and the other epitope for detection. Epitopes are available for N- or C-terminal addition, and, in some cases, three separate vectors are available to accommodate any reading frame. One vector contains a sequence that expresses the same epitope, HTTPHH, irrespective of the reading frame. One vector is available to insert the epitope internally using a multiple cloning site and a restriction enzyme. Some vectors provide

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protease cleavage sites to remove the epitope after protein purification, and others provide nuclear localization signals or secretion signals. For purification of the epitope-tagged protein, systems are available, e.g. FLAG and Protein-C, that allow one-step purifications using native or mildly denaturing conditions. Recently, one company has offered a large number of individual clones expressing pretagged yeast or mammalian proteins. Epitope-tagged molecular weight markers are even available for inclusion in Western blots.


Detecting the Tag Epitope-tagged proteins are detected using the same range of methods that are used with standard protein-specific antibodies: Western blotting, immunoprecipitation, ELISAs, immunofluorescence, immunoelectron microscopy, surface plasmon resonance, etc (33). Because the guest peptides are detected using standardized monoclonal or polyclonal antibodies, once a protocol has been optimized for a particular tagged protein, it can generally be applied to any other similarly tagged protein expressed under the same conditions. Further, because the epitope is precisely known, competition with a peptide representing the epitope can be used to confirm the specificity of the antibody for the tagged protein. Often this is not possible with a standard antibody to the host protein itself, since the epitope or epitopes to which the antibody binds are not known.

SUMMARY The major advantages and limitations of epitope tagging are listed below.

Advantages Tag is rapidly and easily added to a known location in the gene; Multiple tags can be added if necessary; Well-characterized antibodies are used; The antibody is specific to the tag, therefore cross-reaction with related proteins is avoided; Proteins and protein complexes can be purified using standardized gentle conditions; Tagged proteins can be distinguished from otherwise identical untagged proteins; and Immunochemistry is possible for poorly immunogenic proteins.

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Limitations A cloned and characterized gene or cDNA must be available; The epitope tag may interfere with protein structure or function; Generally, epitope-tagged genes are expressed at abnormal levels due to the use of heterologous promoters; and


The epitope-tagged gene must be introduced into the cell, tissue, or organism of interest.

APPLICATIONS Epitope tagging has been used to address a wide variety of experimental questions. In the sections to follow, we cite some recent applications of epitope tagging to studies of protein expression, localization, purification, topology, dynamics, interactions, functional analysis, and discovery. We have made no attempt to be exhaustive in these citations, as there are well over a thousand published studies in which epitope tagging has been utilized. Nor have we attempted, in most cases, to discover and cite the earliest reference to a particular application.

Expression In a straightforward application, epitope tagging can be used to assess whether a gene that is introduced into a cell is expressed and to assess its level of in vivo expression. Coexpression of two genes can be monitored by tagging both of them, with either the same or different tags (42, 57, 78). Comparative expression of mutant and wild-type proteins can also be examined, as when levels of mutant and wild-type ovine Mel1a (beta melatonin receptor) were compared using FLAG-tagged genes (12).

Localization Numerous studies have taken advantage of epitope tagging to determine, or confirm, the subcellular location of a gene product. Immunofluorescence microscopy is the most prevalent means for doing this (e.g. 28, 39, 66). Immunoelectron microscopy has been employed in many studies to determine location at the ultrastructural level (e.g. 21, 108, 116, 117). Localization to particular cellular compartments has also been explored by fractionating cells and examining the fractions for the presence of the tagged protein, for example, by immunoprecipitation followed by Western blots or appropriate biochemical assays (27, 73, 99). Epitope tagging has also been used to investigate which regions of a protein contribute to localization (25, 27, 37, 52, 82, 110, 115).

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Purification An important feature of epitope tagging is that it provides a specific means to purify the tagged protein. Immunoaffinity chromatography (7, 48, 51, 59, 76, 79) and immunoprecipitation (11, 15, 36, 55, 65, 71, 104) are the two most frequently used purification methods. Immunoprecipitation from a variety of cellular compartments has been used to purify many different proteins, including endoplasmic reticulum BiP (70), nuclear RRN5 gene product (45), TAF proteins (84), nuclear pore complex Rat2p (34), bacterial cytoplasmic membrane (92), and photolabeled vasopressin receptor (83). If one desires to remove a terminal tag after purification, a protease recognition site can be included at an appropriate location. Several of the newer expression vectors with epitope-tagging features include such sites. Another option is N-terminal tagging with the FLAG epitope, which contains within it a recognition site for the protease enterokinase. Polyhistidine tags are very popular for purification purposes because the tagged proteins can be purified using immobilized metal affinity chromatography (IMAC) with nickel or cobalt affinity resins. IMAC has distinct advantages over immunoaffinity chromatography for purification because the effective density of the nickel or cobalt in the resin can be much higher than for immobilized antibodies, allowing for greater binding and greater recovery of the tagged protein. The literature on the use of these tags for affinity purification is extensive but is not surveyed here (for a review see 76). The polyhistidine tag can also serve as an epitope tag when probed with anti-His6 antibodies, so that the tagged protein can be examined at the cellular level by immunological methods, as described above. The use of the His-tag as a primary epitope tag has not found great favor yet, perhaps because the available antibodies lack high specificity and low background. Thus, one frequently encounters the tagging of the same protein with both a His-tag for affinity purification and a standard epitope tag for immunodetection.

Topology Integral membrane proteins, including receptors and channels, have been favorite targets for epitope tagging. Visualization of the tag can reveal which parts of the protein are intracellular or extracellular, and/or which parts are exposed on the surface of an isolated organelle. Epitopes are engineered into specific regions of the protein or into a number of randomly selected regions; reaction with the antibody indicates accessibility, thereby suggesting location of the epitope with respect to the membrane. A number of proteins subjected to this type of analysis are listed in Table 2. Others include Na/K ATPase (8), P-glycoproteins (44), NMDA receptor (38), monoamine neurotransmitter transporters (47), and SGLT1 (106).

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Dynamics


Epitope tagging has been used to study the movements of proteins through cellular compartments. Golgi trafficking of GD3 synthase (64) and PKC epsilon (54), and Golgi fragmentation (116) have been monitored. Stress-induced HSF1 granules in the nucleus were discovered using tagged HSF1 (13). Isoform sorting and localization (49, 94) and the roles of alternatively spliced exons containing information for the selectivity of homo- versus heterodimerization (26) have also been explored.

Interactions In a number of studies, coimmunoprecipitation of a protein complex with antitag antibodies has been used to identify and analyze protein-protein interactions (15, 45, 65, 104, 111). The method has assisted in distinguishing dimerization characteristics (14, 35), homomultimer (114), homodimer (118) and heterodimer assembly (19), and heteromultimer assembly (79). Using an in vitro system and c-myc-tagged peptide fragments, an association was demonstrated between transmembrane domains and the association was related to assembly of the Kv1.3 channel (97). A system has been developed using epitope tagging for verifying interactions detected in yeast two-hybrid selections (113). Protein ligands have also been tagged to study interactions with receptors (77).

Functional Analysis Complementation, the classic genetic test for function, has been effectively applied to epitope-tagged genes in many studies (e.g. 10, 31, 43, 96, 99, 101). The epitope-tagged gene is introduced into cells or organisms with a null or temperature-sensitive mutation in the gene of interest; restoration of the wildtype phenotype indicates that the tagged protein is functional in vivo. For most applications of epitope tagging, a detailed functional comparison of the epitope-tagged protein to its wild-type counterpart has not been performed. In some studies, however, the authors have looked closely at the possibly subtle consequences of the addition of the epitope tag to the polypeptide. A stringent testing of FLAG epitope-tagged cystic fibrosis transmembrane conductance regulator (CFTR) showed that control mechanisms for channel gating were not affected (95). Likewise, epitope-tagged tyrosine receptor (TSHR) retained function and glycosylation (102). Receptors have been expressed in heterologous systems and retain function (69). The addition of an epitope to hepatic lipase did not alter the enzyme secretion rate or specific activity (5). SGLT1 is a protein for which there are no high-affinity antibodies so the VSV-G epitope was added to the carboxyl terminus of the protein. The tagged protein was found to localize to the cell surface, to function in transport, and to show sensitivity to specific inhibitors. However, upon rigorous testing of its biochemical properties, it was

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revealed that the tagged protein had an altered glucose transport but not Na+ transport (106). These, like many of the studies cited previously, illustrate that the effects of epitope tagging on the function of a target protein are idiosyncratic and depend on the particular tag and on the particular site in the protein that is tagged. Often the effects are negligible, sometimes they are subtle but significant, and sometimes they are extreme.


Transgenics A number of recent papers describe the analysis of transgenic animals and plants expressing epitope-tagged genes. Such experiments can reveal which tissues and cell types express the protein of interest, as well as indicate where in the cell the protein is localized, as was done for the AHA3 and AHA2 proton pump in Arabidopsis (16, 17). Epitope tagging was used to explore the functions of individual domains in a Drosophila protein, DLG, by tagging individual isolated domains of the protein and expressing them in wild-type or null-mutant transgenic animals (41). Transgenic organisms have been used to explore the trafficking of proteins between tissues and cells, as in the case of transgenic Caenorhabditis carrying an epitope-tagged version of the emb-9 gene, in which it was shown that type IV collagen made in body wall muscle cells can assemble into the pharyngeal, intestinal, and gonadal basement membranes (29). The importance of posttranslational modifications has also been explored in transgenic organisms using epitope tagging, as when a transgene carrying the promoter from the human MARCKS gene and an epitope-tagged human coding sequence was shown to complement MARCKS deficiency in mice, as did a mutant epitope-tagged protein that was incapable of C-terminal myristoylation (101). Epitope tagging has also been used to distinguish between closely related protein isoforms in transgenic organisms, as in the case of transgenic mice in which an epitope-tagged 14-kD myelin basic protein (MBP) isoform was shown to segregate to the plasmalemma preferentially with respect to the three other major MBP isoforms (32).

Gene Therapy Epitope tagging has also been useful in the context of gene therapy, in part because it allows one to distinguish between the native protein and an equivalent, but tagged, protein. In a study of delivery of naked DNA to the myocardium, a plasmid containing an epitope-tagged myosin heavy chain was injected into mouse heart and shown to express the tagged protein in vivo (56). Viral delivery has also been studied using recombinant adenovirus to deliver epitopetagged neurofilament M to transgenic mice lacking axonal neurofilament. It was shown using confocal and electron microscopy that the tagged protein was transported in unpolymerized form along axonal microtubules (103). In another

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investigation involving adenovirus (112), epitope-tagged virions were targeted to endothelial cells using bispecific antibodies that recognized the virions and a specific cell surface receptor.


Functional Genomics and Proteomics We expect that epitope tagging will remain a standard method for detecting, purifying, and analyzing the products of cloned genes for many more years. But the method can also be applied to the discovery of genes and proteins by delivering epitope-encoding sequences to random sites in the genome and monitoring the appearance of epitope-tagged proteins. Approaches of this kind have been described for Chlamydomonas, Drosophila, and Saccharomyces (43, 89), and, with appropriate modifications, they can be used to tag and identify mammalian genes and proteins. Epitope tagging thus has the potential to make major contributions to the emerging fields of functional genomics and proteomics in the years to come. Visit the Annual Reviews home page at http://www.AnnualReviews.org

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Annual Review of Genetics Volume 32, 1998


CONTENTS Alfred D. Hershey, Allan Campbell, Franklin W. Stahl

1

The Role of the FHIT/FRA3B Locus in Cancer , Kay Huebner, Preston N. Garrison, Larry D. Barnes, Carlo M. Croce

7

Regulation of Symbiotic Root Nodule Development, M. Schultze, A. Kondorosi

33

Targeting and Assembly of Periplasmic and Outer-Membrane Proteins in Escherichia coli , Paul N. Danese, Thomas J. Silhavy

59

The Genetics of Breast Cancer Susceptibility, Nazneen Rahman, Michael R. Stratton

95

Nonsegmented Negative Strand RNA Viruses: Genetics and Manipulation of Viral Genomes, Karl-Klaus Conzelmann

123

The Genetics of Disulfide Bond Metabolism, Arne Rietsch, Jonathan Beckwith

163

Comparative DNA Analysis Across Diverse Genomes, Samuel Karlin, Allan M. Campbell, Jan Mrázek

185

The Ethylene Gas Signal Transduction Pathway: A Molecular Perspective, Phoebe R. Johnson, Joseph R. Ecker

227

Molecular Mechanisms of Bacteriocin Evolution, Margaret A. Riley

255

Alternative Splicing of Pre-mRNA: Developmental Consequences and Mechanisms of Regulation, A. Javier Lopez

279

Kinetochores and the Checkpoint Mechanism that Monitors for Defects in the Chromosome Segregation Machinery, Robert V. Skibbens, Philip Hieter

307

The Diverse and Dynamic Structure of Bacterial Genomes, Sherwood Casjens

339

Recombination and Recombination-Dependent DNA Replication in Bacteriophage T4, Gisela Mosig

379

Natural Selection at Major Histocompatibility Complex Loci of Vertebrates, Austin L. Hughes, Meredith Yeager

415

Evolution and Mechanism of Translation in Chloroplasts, Masahiro Sugiura, Tetsuro Hirose, Mamoru Sugita

437

Genetics of Alzheimer's Disease, Donald L. Price, Rudolph E. Tanzi, David R. Borchelt, Sangram S. Sisodia

461

THE CRITICAL ROLE OF CHROMOSOME TRANSLOCATIONS IN HUMAN LEUKEMIAS, Janet D. Rowley

495

Early Patterning of the C. elegans Embryo, Lesilee S. Rose, Kenneth J. Kemphues

521


Genetic Counseling: Clinical and Ethical Challenges, M. B. Mahowald, M. S. Verp, R. R. Anderson

547

Mating-Type Gene Switchingin Saccharomyces cerevisiae , James E. Haber

561

Epitope Tagging, Jonathan W. Jarvik, Cheryl A. Telmer

601

The Leptotene-Zygotene Transition of Meiosis, D. Zickler, N. Kleckner

619