Introduction

Epitope tagging has led to a versatile genetic strategy enabling rapid and effective immune-activity generation of the target proteins against commercial antibodies (1). Two approaches have been used to insert a tag into the target protein. The first approach uses an expression plasmid that already encodes the epitope sequence, while the other employs the PCR-based direct insertion of the epitope encoding sequence into the chromosome loci of the target protein (1,2). The latter approach allows for not only the deletion of target chromosome loci, but also for the insertion of heterologous DNA into the genome with single base pair precision. However, gene disruption and epitope tagging in Escherichia coli using linear double-stranded DNA fragments is inefficient due to the exonuclease activity present in E. coli (3). Recently, the effectiveness of the bacteriophage A. Red recombination system brought a solution to the problem (4). Using the system, researchers have developed a method that uses PCR-based gene disruptions to inactivate chromosomal genes in E. coli (5). Along these same lines, a modified system was designed to generate protein fusions in Salmonella (6) that successfully used immunological methods to detect single epitope-tagged proteins. However, the low detection sensitivity limited its ability to carry out most experimental applications.

Detection sensitivity is one of the most important aspects of epitope tagging due to the inherent low abundance of tagged proteins (e.g., transcription factors) being analyzed. Thus, it is highly desirable to use tandem copies of the epitope tag to improve the signal-to-noise ratio. One of the main concerns of tandem epitope tagging is that the introduction of multiple epitope tags could potentially affect the function of target proteins (1). Most proteins accepted a tag at one or both termini without significantly affecting function, however, it is not known if the introduction of tags elsewhere in the protein affects function. Also, no simple relationship has been established between the size of the tag and how well it is tolerated.

Although a great deal of effort has been devoted to developing efficient tagging systems, what seems to be lacking is a versatile tandem epitope tagging system based on PCR and λ Red-mediated recombination. To address this need, a series of template plasmids carrying tandem epitope sequences have been constructed, and a method has been applied whereby PCR-generated fragments containing the tandem epitope sequence, Flp recombinase target (FRT) sites, and an antibiotic resistance gene, flanked by homologous sequences of the gene of interest, are inserted into the E. coli chromosome. This method of tandem epitope tagging has several advantages. It does not require prior cloning of the gene and can be used to precisely tag proteins within the E. coli chromosome. The Flp recombinase allows for the removal of the antibiotic resistance gene from the inserted heterologous PCR fragment once the correct orientation is confirmed. The use of tandem tags potentially enables use in more stringent experimental conditions to increase the signal-to-noise ratio in further experimental applications. Another significant advantage of this tagging method is the library of constructed template plasmids, which provides a resource allowing for the rapid construction of an array of tandem-tagged proteins that can be tested for functionality.

Materials and Methods

Construction of Template Plasmids

E. coli strains were obtained from E. coli Genetic Stock Center (cgsc.biology.yale.edu). Complementary oligonucleotide primers of HA tag and myc tag encoding sequence were designed to hybridize to each other at the 3′ end of 12 bases and encode two units of epitope sequence (7). One nanomole of each primer was mixed and heated at 65°C for 2 min. The primer mixtures were then phosphorylated using 40 U T4 polynucleotide kinase (Stratagene, La Jolla, CA, USA) at 37°C for 1 h. After the enzyme was inactivated at 70°C for 10 min, the mixture was cooled to room temperature. The crossover PCR was performed using 1.5 µM phosphorylated oligonucleotide primer mixture in a final volume of 100 µL containing 5 U Stoffel fragment of Taq DNA polymerase (Perkin Elmer, Wellesley, MA, USA). PCR amplification conditions were 30 cycles with 30 s denatur-ation at 94°C, 30 s annealing at 60°C, and 30 s extension at 72°C. The PCR product was purified and inserted into Novagen® pSTBlue-1 (EMD Biosciences, Madison, WI, USA) using blunt-end ligation and confirmed by DNA sequencing. A DNA fragment, which contained the flanking FRT sites and kanamycin resistance gene, was amplified from pKD13 (5) using primers carrying oligonucleotide extensions with HindIII and SacI restriction sites. The PCR product was cleaved by HindIII/SacI and then ligated into the HindIII/SacI cleaved pSTBlue-1:(n)-Myc or pSTBlue-1:(n)-HA to obtain the pBOP5 and pBOP6 series, respectively.