M o l e c u l a r B i o l o g y
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
. 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
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 BAPB.1x FLAG BAP12345
Figure 4. Light microscopy of immunostained cells.
(A) COS-7 cells transfected with p3xFLAG-CMV-7-BAP.(B) Control COS-7 cells.