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) |
|
){kind=link}
){kind=link}
){kind=link}
){kind=link}
){kind=link}