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3x FLAG: Ultra-Sensitive Detection of Recombinant Proteins

by Ron Hernan, Ken Heuermann and Bill Brizzard

Sigma-Aldrich Corporation, St. Louis, MO, USA

IntroductionEpitope tagging has become a powerful tool for thedetection and purification of expressed proteins. Thismethodology has been used for protein localization,immunoprecipitation, and protein-protein interaction.Many types of tags have been used, with c-myc andFLAG® being two of the most popular epitope tagsutilized.1 Generally, these sequences are fused to the N-or C-terminus of the expressed protein making them moreaccessible for antibody detection and less likely to causesevere structural or functional perturbations.

The original FLAG sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, is recognized by two monoclonal antibodies, M1 andM2.2,3 In addition, the FLAG sequence with an initiatormethionine attached is recognized by the M2 antibodyand a third antibody, M5.4 The last five amino acids of theFLAG sequence are the recognition site for the proteaseenterokinase, thus, allowing for removal of the FLAGepitope tag.

The FLAG tag has been used in expression systems fordetection and purification of heterologous proteins in E. coli,5 Saccharomyces cerevisiae,3,6 Drosophila,7

Baculovirus,8,9 and mammalian systems.10,11 Formammalian expression systems, expression levels are lowand effective detection of expressed foreign proteins usingestablished methods can be difficult. We describe amammalian expression plasmid containing multiple FLAGepitopes in tandem, p3x FLAG CMV-7, designed forintracellular expression with increased sensitivity ofdetection. This vector contains the cytomegalovirus (CMV)promoter12 and simian virus 40 (SV40) origin of replicationfor efficient expression in COS-7 cells.13 We compare thedetection of triple FLAG-tagged bacterial alkalinephosphatase (BAP) expressed and purified from E. coli tosingle FLAG-tagged BAP. We demonstrate the efficacy ofpFLAG-CMV-7 as a mammalian expression vector.

Materials and MethodsAll materials were supplied by Sigma Chemical (St. Louis, MO)unless otherwise stated.

Vector ConstructionWe have constructed a vector for expression of proteins inmammalian host cells using a modified version of theFLAG expression system, which contains 3x FLAGsequences in tandem (Figure 1).

Expression of Bacterial Alkaline Phosphatase in E. coliTo address whether a triple FLAG fusion protein producesa more sensitive response than the traditional FLAG

epitope tag, a triple FLAG version of bacterial alkalinephosphatase was constructed for expression in E. coli(Figure 2). The vector p3x FLAG-ATS-BAP was transformedinto E. coli and the 3x FLAG-BAP protein was expressedand purified. In addition, an N-FLAG-BAP proteincontaining the traditional epitope tag was also expressedand purified.5

Figure 1. p3xFLAG-CMV-7 expression vector. (A) Plasmid map of the p3xFLAG-CMV-7 showing the CMV promoter, human growth hormonetranscription termination and polyadenylation site, SV40 origin of replication,Col E1 origin of replication, and ß-lactamase gene. (B) DNA and proteinsequence of 3xFLAG-CMV-7 multiple cloning site and FLAG sequences.

Figure 2. Expression vector p3xFLAG-ATS-BAP. Vector map of p3xFLAG-ATS-BAP showing insertion of the phoA coding region into pFLAG-ATS-BAP.

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Western BlotPurified 3x FLAG-BAP and N-FLAG-BAP were diluted with2x Laemlli buffer,10 boiled for five minutes and then placedon ice. Samples were resolved on a 15% SDS-PAGE usingthe method of Laemlli10 and then transferred tonitrocellulose membranes. The membrane was blockedwith phosphate buffered saline containing 3% non-fat drymilk for 1 hour and then rinsed three times in TBS, 0.05%Tween® 20 (TBS-T). The membrane was incubated withM2 antibody at a final concentration of 10 µg/ml for 30minutes in TBS-T and then rinsed three times in TBS-T.The membrane was then incubated for 30 minutes with agoat anti-rabbit IgG (whole molecule) horseradishperoxidase (HRP) conjugate, diluted 1:10,000 in TBS-T,then rinsed three times in TBS-T. The FLAG-taggedproteins were detected with the HRP conjugate andvisualized by chemilumenescent detection using ECL™ kit(Amersham Pharmacia Biotech, Piscataway, NJ ) withexposures from 1-30 minutes on Kodak X-Omat® MR film(Eastman Kodak, Rochester, NY).

Expression of Bacterial Alkaline Phosphatase in Mammalian CellsFunctionality of the pFLAG-CMV-7 mammalian vector was demonstrated by transfection of COS-7 cells withpFLAG-CMV-7-BAP and detection of BAP expression byimmunostaining. At 48 hours post-transfection cells werefixed with methanol:acetone, washed, and incubated for 1 hour with 10 µg/ml M2 monoclonal antibody:HRPconjugate in TBS. Cells were then washed and stainedwith 100 µg/ml o-dianisidine in the presence of 0.015%hydrogen peroxide, in TBS. Cells not stained with o-dianisidine were counter-stained for 1 minute using a1:1 solution of Mayer's hematoxylin in water.

ResultsThis vector construct was designed to improve thedetection limit of expressed proteins in mammalian hostcells. The first two flag FLAG peptides are modificationsof the original FLAG sequences previously described: Asp-Tyr-Lys-Asp-His-Asp. A Gly-Ile spacer joins the sequences.These alternative sequences arise from phage displaystudies in which a different binding motif wasdetermined.14 This allows the introduction of additionalFLAG antibody binding sites without the addition of extraenterokinase recognition/cleavage sites.

The p3x FLAG-CMV-7 expression vector contains thepromoter region of the human cytomegalovirus majorimmediate early gene, which allows for constitutiveexpression of cloned genes in mammalian cell lines. TheKozak consensus sequence15 is provided in the vectoralong with a multiple cloning site, which allows for avariety of cloning strategies. The multiple cloning site iscompatible with the other existing CMV mammalianexpression vectors. In addition, the expression vectorcontains the SV40 origin of replication for efficient high-level transient expression13 and a DNA segment from thehuman growth hormone containing transcriptionaltermination sequence and polyadenylation signals.16

p3x FLAG-CMV-7 contains the ß-lactamase gene forselection of the plasmid in E. coli. Using this vector, wehave successfully transfected and expressed heterologousproteins in COS cells.

Bacterial Alkaline Phosphatase ExpressionComparison of the sensitivity of the single FLAG-BAP

versus the triple FLAG-BAP was demonstrated by Westernblot analysis as previously described. Figure 3 shows theWestern blot of purified single FLAG-BAP and triple flagFLAG-BAP probed with ANTI-FLAG®-M2 monoclonalantibody and detected by chemiluminescence. The resultsclearly indicate a 10-fold increase in detection limit of thetriple FLAG-BAP compared to the single FLAG-BAP fusionprotein. We were able to detect 500 picograms ofpurified 3x FLAG- BAP with exposures as short as 1 minute. With increased exposure time, detection as lowas 100 picograms has been achieved but with increasedbackground (data not shown).

COS-7 cells expressing FLAG-tagged BAP fusion proteinare shown in Figures 4(A) and (B). Detection oftransfected cells is typically observed within 10 minutes ofadding o-dianisidine.

DiscussionThe FLAG epitope tag has been effectively used to detectand purify proteins5 in mammalian and bacterial systems.We have demonstrated that the presence of three FLAGepitopes greatly increases the detection limit of purifiedbacterial alkaline phosphatase. Moreover, we have foundthat 3x FLAG-BAP cannot be eluted from ANTI-FLAG M2affinity gel by competition with the original FLAG peptide.However, 3x FLAG-BAP and the 1x FLAG-BAP can becompetitively eluted from the ANTI-FLAG M2 affinity gelusing 3x FLAG peptide (data not shown). The p3x FLAG-CMV-7 vector was designed for expression and detection of heterologous proteins in mammalian cellsand is compatible with existing pFLAG-CMV vectors. Thisallows for easy subcloning between vectors containing thesingle FLAG and the triple FLAG. The immunostainingresults show that expression of the phoA gene, which

Figure 3. Detection of purified 3xFLAG-BAP by Western blot. (A) Westernblot of purified 3xFLAG-BAP using ANTI-FLAG M2 antibody (Lane 1, 0.5 ng;Lane 2, 1.0 ng; Lane 3, 2.0 ng; Lane 4, 5.0 ng; and Lane 5, 10 ng). (B) Westernblot of purified N-FLAG-BAP using ANTI-FLAG M2 antibody (Lane 1, 0.5 ng;Lane 2, 1.0 ng; Lane 3, 2.0 ng; Lane 4, 5.0 ng; and Lane 5, 10 ng).

A. 3x FLAG BAP

B. 1x FLAG BAP

1 2 3 4 5

Figure 4. Lightmicroscopy ofimmunostained cells.(A) COS-7 cells transfectedwith p3xFLAG-CMV-7-BAP.(B) Control COS-7 cells.

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codes for BAP in COS-7 cells, is not significantly perturbedby addition of the 3x FLAG sequence.

The M2 antibody reacts with the alternate epitope in the3x FLAG sequence. In contrast, M5 antibody fails to showthe increased sensitivity that the M2 antibodydemonstrates, (results not shown). Recent results usingphage display17 have demonstrated that the criticalresidues for M2 binding and M5 binding are slightlydifferent. M2 antibody prefers the sequence Asp-Tyr-Lys-Xxx-Xxx-Asp-Xxx-Xxx, while M5 prefers Asp-Tyr-Xxx-Xxx-Asp-Asp-Xxx-Xxx. The triple FLAG sequence Asp-Tyr-Lys-Asp-His-Asp clearly favors the binding of M2 over that ofM5 or M1 antibodies. This expression system allows forincreased sensitivity and detection of the FLAG epitopetagging system while retaining the benefits of the FLAGmammalian expression system. These include FLAG’shydrophilic nature, small size, and the ability to removethe FLAG tag using enterokinase.

References 1. Evan, G., et al., Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol., 5, 3610-3616 (1985).

2. Hopp, T., et al., A short polypeptide marker sequence useful forrecombinant protein identification and purification. BioTechnology, 6, 1204-1210 (1988).

3. Prickett, K., et al., A calcium dependent antibody for identification andpurification of recombinant proteins. BioTechniques, 7, 580-589 (1989).

4. Brizzard, B., and Chubet, R., in Current Protocols in Neuroscience. Crawley,J. N., et al., Eds, pp. 5.8.1-5.8.11 (John Wiley & Sons, New York, N.Y., 1999).

5. Brizzard, B., et al. Immunoaffinity purification of FLAG epitope-taggedbacterial alkaline phosphatase using a novel monoclonal antibody and peptideelution. BioTechniques, 16, 730-735 (1984).

6. Lee, J., et al. A protein kinase involved in the regulation of inflammatorycytokine biosynthesis. Nature, 372, 739-746 (1994).

7. Xu, T., and Rubin, G. Analysis of genetic mosaics in developing and adultDrosophila tissues. Development, 117, 1223-1237 (1993).

8. Dent, P., et al., Regulation of raf-1 and raf-1 mutants by ras-dependent andras-independent mechanisms in vitro. Mol.Cell Biol., 15, 4125-4135 (1995).

9. Ritchie, P., et al. Baculovirus expression and biochemical characterization ofthe human microsomal triglyceride transfer protein. Biochem. J., 338, 305-310(1999).

10. Schulte am Esch II, J. et al., Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity. Biochemistry, 38, 2248-2258 (1999).

11. Overholt, S., et al., Head and neck squamous cell growth suppressionusing adenovirus-p53-FLAG: a potential marker for gene therapy trials. Clin.Cancer Res., 3, 185-191 (1997).

12. Thomsen, D., et al., Promoter-regulatory region of the major immediateearly gene of human cytomegalovirus. Proc. Natl. Acad. Sci. USA, 81, 659-663 (1984).

13. Okayama, H., and Berg, P., A cDNA cloning vector that permits expressionof cDNA inserts in mammalian cells. Mol. Cell. Biol., 3, 280-289 (1983).

14. Miceli, R., et al., Two-stage selection of sequences from a random phagedisplay library delineates both core residues and permitted structural rangewithin an epitope. J. Immunol. Methods, 167, 279-287 (1994).

15. Kozak, M., Point mutations close to the AUG initiator codon affect theefficiency of translation of rat preproinsulin in vivo. Nature, 308, 241-246(1984).

16. Seeburg, P., The human growth hormone gene family: nucleotidesequences show recent divergence and predict a new polypeptide hormone.DNA, 1, 239-249 (1982).

17. Slootstra, J., et al., Identification of new tag sequences with differentialand selective recognition properties for the anti-FLAG monoclonal M1, M2and M5. Molecular Diversity, 2, 156-164 (1996).

Adapted from Biotechniques 28, 789-793 (April 2000) bypermission. Figures 1 (p. 790) and 2 (p. 791), adapted.

FLAG and ANTI-FLAG are registered trademarks of the Sigma-AldrichCorporation. Tween is a registered trademark of ICI. ECL is a trademark of Amersham Pharmacia Biotech. X-Omat is a registered trademark ofEastman Kodak.

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About the AuthorsRon Hernan, Ph.D., and Ken Heuermann, M.S., are senior scientists in Recombinant Protein Expression R&D at Sigma-Aldrich,St. Louis, MO. Bill Brizzard, Ph.D., is the manager of Technology Transfer at Sigma-Aldrich, St. Louis, MO.

ORDERING INFORMATIONProduct Code Product Name Unit Price

E 2400 p3x FLAG-CMV™-7 Expression Vector 20 µg $238.60E 4026 p3x FLAG-CMV™-7.1 Expression Vector 20 µg $250.00E 4151 p3x FLAG-CMV™-8 Expression Vector 20 µg $250.00E 4276 p3x FLAG-CMV™-9 Expression Vector 20 µg $250.00E 4401 p3x FLAG-CMV™-10 Expression Vector 20 µg $250.00E 4776 p3x FLAG-CMV™-13 Expression Vector 20 µg $250.00E 4901 p3x FLAG-CMV™-14 Expression Vector 20 µg $250.00*All p3xFLAG vectors include N-terminal p3xFLAG-CMV-BAP as control.

F 4799 3x FLAG peptide 4 mg $152.3025 mg $710.50

P 2104 N-TERMINAL 3x FLAG-BAP 0.1 mg $130.00

Product Code Product Name Unit Price

A 1205 ANTI-FLAG M2 Affinity Gel 1 ml $227.905 ml $481.0010 ml $961.9025 ml 1359.70

F 3165 ANTI-FLAG M2 Antibody 0.2 mg $136.301 mg $232.805 mg $384.00

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