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Cloning and Expression - FLAG and 3xFLAG Overview

Cloning and Expression - FLAG and 3xFLAG Overview



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Published by Sigma-Aldrich
An overview on the FLAG and 3xFLAG for Detection and Purification of Proteins, from Sigma-Aldrich.
An overview on the FLAG and 3xFLAG for Detection and Purification of Proteins, from Sigma-Aldrich.

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Categories:Types, Brochures
Published by: Sigma-Aldrich on Feb 14, 2009
Copyright:Attribution Non-commercial


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Order: 1.800.325.3010 Technical Service: 1.800.325.5832www.sigma-aldrich.com
3x FLAG:Ultra-Sensitive Detectionof Recombinant Proteins
by Ron Hernan, Ken Heuermann and Bill Brizzard 
Sigma-Aldrich Corporation, St. Louis, MO, USA
Epitope 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-
being two of the most popular epitope tagsutilized.
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.
In addition, the FLAG sequence with an initiatormethionine attached is recognized by the M2 antibodyand a third antibody, M5.
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 
Saccharomyces cerevisiae
and mammalian systems.
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)promoter
and simian virus 40 (SV40) origin of replicationfor efficient expression in COS-7 cells.
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 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 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. coli 
To address whether a triple FLAG fusion protein producesa more sensitive response than the traditional FLAGepitope 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.
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 protein sequence 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.
    M   o    l   e   c   u    l   a   r    B    i   o    l   o   g   y
    M   o    l   e   c   u    l   a   r    B    i   o    l   o   g   y
Order: 1.800.325.3010 Technical Service: 1.800.325.5832www.sigma-aldrich.com
Western Blot 
Purified 3x FLAG-BAP and N-FLAG-BAP were diluted with2x Laemlli buffer,
boiled for five minutes and then placedon ice. Samples were resolved on a 15% SDS-PAGE usingthe method of Laemlli
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 Phosphatasein Mammalian Cells
Functionality of the pFLAG-CMV-7 mammalian vectorwas 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 for1 hour with 10 µg/ml M2 monoclonal antibody:HRPconjugate in TBS. Cells were then washed and stainedwith 100 µg/ml
-dianisidine in the presence of 0.015%hydrogen peroxide, in TBS. Cells not stained with
-dianisidine were counter-stained for 1 minute using a1:1 solution of Mayer's hematoxylin in water.
This 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.
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 sequence
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 expression
and a DNA segment from thehuman growth hormone containing transcriptionaltermination sequence and polyadenylation signals.
p3x FLAG-CMV-7 contains the
-lactamase gene forselection of the plasmid in
E. coli 
. Using this vector, wehave successfully transfected and expressed heterologousproteins in COS cells.
Bacterial Alkaline Phosphatase Expression
Comparison of the sensitivity of the single FLAG-BAPversus 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 as1 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
The FLAG epitope tag has been effectively used to detectand purify proteins
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). Thep3x FLAG-CMV-7 vector was designed for expressionand 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
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).
Figure 4. Light microscopy of immunostained cells.
(A) COS-7 cells transfected with p3xFLAG-CMV-7-BAP.(B) Control COS-7 cells.

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