Life Science Quarterly July 2000

July 2000 Feature Article

 Life Science
Quarterly
 A TECHNICAL APPLICATION NEWSLETTER VOL 1    July 2000

 Molecular Biology Feature Article


3x FLAG: Ultra-Sensitive Detection of Recombinant Proteins

by Ron Hernan, Ken Heuermann and Bill Brizzard
Sigma-Aldrich Corporation, St. Louis, MO, USA.

Introduction

Epitope tagging has become a powerful tool for the detection and purification of expressed proteins. This methodology has been used for protein localization, immunoprecipitation, and protein-protein interaction. Many types of tags have been used, with c-myc and FLAG® being two of the most popular epitope tags utilized.1 Generally, these sequences are fused to the N- or C-terminus of the expressed protein making them more accessible for antibody detection and less likely to cause severe structural or functional perturbations.

The original FLAG sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, is recognized by two monoclonal antibodies, M1 and M2.2,3 In addition, the FLAG sequence with an initiator methionine attached is recognized by the M2 antibody and a third antibody, M5.4 The last five amino acids of the FLAG sequence are the recognition site for the protease enterokinase, thus, allowing for removal of the FLAG epitope tag. 

The FLAG tag has been used in expression systems for detection and purification of heterologous proteins in E. coli,5 Saccharomyces cerevisiae,3,6 Drosophila,7 Baculovirus,8,9 and mammalian systems.10,11 For mammalian expression systems, expression levels are low and effective detection of expressed foreign proteins using established methods can be difficult. We describe a mammalian expression plasmid containing multiple FLAG epitopes in tandem, p3x FLAG CMV-7, designed for intracellular expression with increased sensitivity of detection. This vector contains the cytomegalovirus (CMV) promoter12 and simian virus 40 (SV40) origin of replication for efficient expression in COS-7 cells.13 We compare the detection of triple FLAG-tagged bacterial alkaline phosphatase (BAP) expressed and purified from E. coli to single FLAG-tagged BAP. We demonstrate the efficacy of pFLAG-CMV-7 as a mammalian expression vector.

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

Vector Construction
We have constructed a vector for expression of proteins in mammalian host cells using a modified version of the FLAG expression system, which contains 3x FLAG sequences in tandem (Figure 1a & Figure 1b).

Expression of Bacterial
Alkaline Phosphatase in E. coli
To address whether a triple FLAG fusion protein produces a more sensitive response than the traditional FLAG epitope tag, a triple FLAG version of bacterial alkaline phosphatase was constructed for expression in E. coli (Figure 2). The vector p3x FLAG-ATS-BAP was transformed into E. coli and the 3x FLAG-BAP protein was expressed and purified. In addition, an N-FLAG-BAP protein containing the traditional epitope tag was also expressed and purified.5

Western Blot
Purified 3x FLAG-BAP and N-FLAG-BAP were diluted with 2x Laemlli buffer,10 boiled for five minutes and then placed on ice. Samples were resolved on a 15% SDS-PAGE using the method of Laemlli10 and then transferred to nitrocellulose membranes. The membrane was blocked with phosphate buffered saline containing 3% non-fat dry milk for 1 hour and then rinsed three times in TBS, 0.05% Tween® 20 (TBS-T). The membrane was incubated with M2 antibody at a final concentration of 10 µg/ml for 30 minutes in TBS-T and then rinsed three times in TBS-T. The membrane was then incubated for 30 minutes with a goat anti-rabbit IgG (whole molecule) horseradish peroxidase (HRP) conjugate, diluted 1:10,000 in TBS-T, then rinsed three times in TBS-T. The FLAG-tagged proteins were detected with the HRP conjugate and visualized by chemilumenescent detection using ECLTM kit (Amersham Pharmacia Biotech, Piscataway, NJ ) with exposures from 1-30 minutes on Kodak X-Omat® MR film (Eastman Kodak, Rochester, NY).

Expression of Bacterial Alkaline Phosphatase
in Mammalian Cells
Functionality of the pFLAG-CMV-7 mammalian vector was demonstrated by transfection of COS-7 cells with pFLAG-CMV-7-BAP and detection of BAP expression by immunostaining. At 48 hours post-transfection cells were fixed with methanol:acetone, washed, and incubated for 1 hour with 10 µg/ml M2 monoclonal antibody:HRP conjugate in TBS. Cells were then washed and stained with 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 a 1:1 solution of Mayer's hematoxylin in water.

Results
This vector construct was designed to improve the detection limit of expressed proteins in mammalian host cells. The first two flag FLAG peptides are modifications of 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 display studies in which a different binding motif was determined.14 This allows the introduction of additional FLAG antibody binding sites without the addition of extra enterokinase recognition/cleavage sites.

The p3x FLAG-CMV-7 expression vector contains the promoter region of the human cytomegalovirus major immediate early gene, which allows for constitutive expression of cloned genes in mammalian cell lines. The Kozak consensus sequence15 is provided in the vector along with a multiple cloning site, which allows for a variety of cloning strategies. The multiple cloning site is compatible with the other existing CMV mammalian expression vectors. In addition, the expression vector contains the SV40 origin of replication for efficient high-level transient expression13 and a DNA segment from the human growth hormone containing transcriptional termination sequence and polyadenylation signals.16 p3x FLAG-CMV-7 contains the ß-lactamase gene for selection of the plasmid in E. coli. Using this vector, we have successfully transfected and expressed heterologous proteins in COS cells.

Bacterial Alkaline Phosphatase Expression
Comparison of the sensitivity of the single FLAG-BAP versus the triple FLAG-BAP was demonstrated by Western blot analysis as previously described. Figure 3 shows the Western blot of purified single FLAG-BAP and triple flag FLAG-BAP probed with ANTI-FLAG®-M2 monoclonal antibody and detected by chemiluminescence. The results clearly indicate a 10-fold increase in detection limit of the triple FLAG-BAP compared to the single FLAG-BAP fusion protein. We were able to detect 500 picograms of purified 3x FLAG- BAP with exposures as short as 1 minute. With increased exposure time, detection as low as 100 picograms has been achieved but with increased background (data not shown).

COS-7 cells expressing FLAG-tagged BAP fusion protein are shown in Figure 4. Detection of transfected cells is typically observed within 10 minutes of adding o-dianisidine.

Discussion
The FLAG epitope tag has been effectively used to detect and purify proteins5 in mammalian and bacterial systems. We have demonstrated that the presence of three FLAG epitopes greatly increases the detection limit of purified bacterial alkaline phosphatase. Moreover, we have found that 3x FLAG-BAP cannot be eluted from ANTI-FLAG M2 affinity gel by competition with the original FLAG peptide. However, 3x FLAG-BAP and the 1x FLAG-BAP can be competitively eluted from the ANTI-FLAG M2 affinity gel using 3x FLAG peptide (data not shown). The p3x FLAG-CMV-7 vector was designed for expression and detection of heterologous proteins in mammalian cells and is compatible with existing pFLAG-CMV vectors. This allows for easy subcloning between vectors containing the single FLAG and the triple FLAG. The immunostaining results show that expression of the phoA gene, which codes for BAP in COS-7 cells, is not significantly perturbed by addition of the 3x FLAG sequence.

The M2 antibody reacts with the alternate epitope in the 3x FLAG sequence. In contrast, M5 antibody fails to show the increased sensitivity that the M2 antibody demonstrates, (results not shown). Recent results using phage display17 have demonstrated that the critical residues for M2 binding and M5 binding are slightly different. 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 of M5 or M1 antibodies. This expression system allows for increased sensitivity and detection of the FLAG epitope tagging system while retaining the benefits of the FLAG mammalian expression system. These include FLAG's hydrophilic nature, small size, and the ability to remove the 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 for recombinant protein identification and purification. BioTechnology, 6, 1204-1210 (1988).
3. Prickett, K., et al., A calcium dependent antibody for identification and purification 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-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. BioTechniques, 16, 730-735 (1984).
6. Lee, J., et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature, 372, 739-746 (1994).
7. Xu, T., and Rubin, G. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development, 117, 1223-1237 (1993).
8. Dent, P., et al., Regulation of raf-1 and raf-1 mutants by ras-dependent and ras-independent mechanisms in vitro. Mol.Cell Biol., 15, 4125-4135 (1995).
9. Ritchie, P., et al. Baculovirus expression and biochemical characterization of the 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 suppression using 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 immediate early 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 expression of 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 phage display library delineates both core residues and permitted structural range within an epitope. J. Immunol. Methods, 167, 279-287 (1994).
15. Kozak, M., Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature, 308, 241-246 (1984).
16. Seeburg, P., The human growth hormone gene family: nucleotide sequences 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 differential and selective recognition properties for the anti-FLAG monoclonal M1, M2 and M5. Molecular Diversity, 2, 156-164 (1996).

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

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

About the Authors

Ron 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 INFORMATION
Product Code  Product Name Unit
E2400 p3x FLAG-CMVTM-7 Expression Vector 20 µg
E4026 p3x FLAG-CMVTM-7.1 Expression Vector 20 µg
E4151 p3x FLAG-CMVTM-8 Expression Vector 20 µg
E4276 p3x FLAG-CMVTM-9 Expression Vector 20 µg
E4401 p3x FLAG-CMVTM-10 Expression Vector 20 µg
E4776 p3x FLAG-CMVTM-13 Expression Vector 20 µg
E4901 p3x FLAG-CMVTM-14 Expression Vector 20 µg
*All p3xFLAG vectors include N-terminal p3xFLAG-CMV-BAP as control.
F4799 3x FLAG peptide  4 mg
    25 mg 
P2104 N-TERMINAL 3x FLAG-BAP 0.1 mg 
RELATED PRODUCTS
Product Code  Product Name Unit
A2220 ANTI-FLAG M2 Affinity Gel 1 ml
    5 ml 
    10 ml 
    25 ml 
F3165 ANTI-FLAG M2 Antibody 0.2 mg
    1 mg
    5 mg 
SUPPORTING LITERATURE
Recombinant Protein Expression booklet (BQW)


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