Multiple epitope tagging of expressed proteins for enhanced detection

In the 3xFLAG tag, the first two of the three tandem FLAG epitopes are the modified sequence Asp-Tyr-Lys-Asp-His-Asp (with a Gly and an Ile spacer between the sequences) instead of the original FLAG Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. This modified motif comes from a phage display study and adds anti-FLAG antibody binding sites without adding extra enterokinase recognition and cleavage sites.

A bacterial alkaline phosphatase (BAP) carrying three tandem FLAG epitopes (3XFLAG-BAP) is detected at a tenfold lower amount than BAP carrying the traditional single FLAG epitope (N-FLAG-BAP) in a western blot of purified protein probed with anti-FLAG M2 antibody and read out by chemiluminescence. Purified 3XFLAG-BAP at 500 pg is detected with exposures as short as 1 min.

The original FLAG epitope Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys is recognized by the anti-FLAG monoclonal antibodies M1 and M2, and the same sequence with an initiator methionine attached is recognized by M2 and M5. The last five amino acids of FLAG, Asp-Asp-Asp-Asp-Lys, are the recognition site of the protease enterokinase, which allows the FLAG epitope to be removed.

The anti-FLAG M2 antibody binds the Asp-Tyr-Lys-Asp-His-Asp repeats of the 3xFLAG tag. Phage display studies indicate that M2 prefers the sequence Asp-Tyr-Lys-Xxx-Xxx-Asp-Xxx-Xxx and M5 prefers Asp-Tyr-Xxx-Xxx-Asp-Asp-Xxx-Xxx, so the 3xFLAG sequence Asp-Tyr-Lys-Asp-His-Asp favors M2 binding over M5 or M1 binding.

3XFLAG-BAP expressed in E. coli BL21(DE3) is captured on M2 antibody-conjugated sepharose 4B in 50 mM Tris-HCl pH 8.0 with 150 mM NaCl and is eluted with 0.1 M glycine pH 3.5.

The mammalian vector p3XFLAG-CMV-7, derived from pCMV-5, contains the human cytomegalovirus major immediate early promoter for constitutive expression, a Kozak consensus sequence, a multiple cloning site, the SV40 origin of replication for high-level transient expression in COS-7 cells, a human growth hormone gene segment providing the transcription termination sequence and polyadenylation signal, and the beta-lactamase gene for selection in E. coli. Its multiple cloning site is compatible with other existing CMV mammalian expression vectors including pFLAG-CMV vectors, allowing subcloning between single-FLAG and triple-FLAG vectors.

A modified E. coli phoA gene (alkaline phosphatase lacking its leader sequence and the first four amino acids of the mature enzyme) cloned into p3XFLAG-CMV-7 as p3XFLAG-CMV-7-BAP gives a 3XFLAG-BAP that is detected in transfected COS-7 cells (2 micrograms of DNA per 35 mm plate, Lipofectamine) by immunostaining with an anti-FLAG M2-HRP conjugate at 72 h after transfection. Expression of the phoA gene in COS-7 cells is not significantly perturbed by addition of the 3XFLAG sequence.