The 3xFLAG® system is an improvement upon the original system by fusing three tandem FLAG® epitopes for a total of 22 amino acids (Figure 1). Detection of fusion proteins containing 3xFLAG® is enhanced up to 200 times more than any other system. Like the original FLAG tag, 3xFLAG® is hydrophilic, contains an Enterokinase cleavage site, and is relatively small. Therefore, the risk of altering protein function, blocking other epitopes, or decreasing solubility is minimized. In cases of low-level expression, 3xFLAG® is ideal. Our selection of CMV vectors provides options for transient or stable expression of 3xFLAG® fusion proteins.
Figure 1.3xFLAG® Sequence. Removal of N-terminal 3xFLAG® tags is possible using enterokinase, which cleaves following the Asp-Asp-Asp-Asp-Lys amino acid sequence at the c-terminal end of the tag
Figure 2.Western Blot of Original FLAG Versus 3xFLAG®. Western blot of purified 3xFLAG®-BAP (bacterial alkaline phosphatase) and FLAG®-BAP transferred onto a nitrocellulose membrane. Detection was performed with ANTI-FLAG® M2 monoclonal primary antibody, anti-mouse-HRP secondary antibody, and ECL™ chemiluminescent substrate.
Figure 3.Comparative Sensitivity of 3xFLAG®. Each protein fusion tag was cloned separately on the C terminal of GST. The purified proteins were diluted from 1 ug to 0.01 ng and analyzed. The limit of detection of each tag was determined by Western blot analysis using the recommended dilutions of each respective primary antibody and secondary anti-mouse IgG-HRP conjugate. The blot was developed using ECL.
Our mammalian expression vectors contain the strong CMV promoter for high-level constitutive expression in mammalian cells. The strong human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mG/L in COS cells. For less potent cell lines, protein levels are typically ~0.1 mG/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication permissive COS cells. Vectors containing the preprotrypsin leader (PPT) sequence direct secretion of FLAG fusion proteins into the culture medium for purification using ANTI-FLAG antibodies, resins, and plates.
Figure 4.MCS Regions
Figure 5.Stable Expression Vector
Figure 6.Bicep Stable Expression Vector