Cloning & Expression

Transient Expression

 CMV Vectors for High Level Mammalian Expression

  • FLAG® and 3xFLAG® vectors for highly sensitive detection and purification using ANTI-FLAG® antibodies, resins, and plates
  • MAT™ vectors for high quality purification using HIS-Select™ resins and 96-well plates
  • N- or C- terminal fusions
  • Dual tag configurations
  • Cytoplasmic expression or secretion
  • Enterokinase removal of N-terminal FLAG tags
  • Expression kits available

 

 

 

Vector Selection Guide

Mammalian Expression Vectors
Transient Expression
  Tag Location Tag Product No. Description
Secretion
Single Tag N-Terminal 3xFLAG E9908 p3xFLAG-CMV 8
Dual Tag N,C-Terminal FLAG and c-myc E8658 pFLAG-Myc-CMV 19
Dual Tag N,C-Terminal 3xFLAG and c-myc E9158 pFLAG-Myc-CMV 23
Cytoplasmic Expression
Single Tag N-Terminal FLAG E7033 pFLAG-CMV 2
Single Tag N-Terminal FLAG E2275 pFLAG-CMV 6a, b, c (set)
Single Tag C-Terminal FLAG E6908 pFLAG-CMV 5.1
Single Tag C-Terminal FLAG E3762 pFLAG-CMV 5a, b, c (set)
Single Tag N-Terminal 3xFLAG E7533 p3xFLAG-CMV 7.1
Dual Tag N,C-Terminal FLAG and c-myc E8783 pFLAG-CMV 20
Dual Tag N,C-Terminal 3xFLAG and c-myc E9283 p3xFLAG-Myc-CMV 24
Dual Tag N,C-Terminal FLAG and MAT C5864 pCMV-FLAG-MAT 1
Dual Tag C-Terminal FLAG and MAT C6114 pCMV-FLAG-MAT 2
Bicistronic
Single Tag N-Terminal FLAG E7029 pBICEP-CMV 3
Dual Tag N-Terminal FLAG and c-myc E9509 pBICEP-CMV 4

We also provide BICEP bicistronic expression vectors for transient expression of two targets. Stable expression vectors may also be used for transient expression.

 

Product MCS Region
E7033, pFLAG-CMV™-2
N-terminal Met-FLAG
E6908,
C-terminal FLAG
E3762, pFLAG-CMV™-5a,b,c
C-terminal FLAG (MCS in all three reading frames)
E2275, pFLAG-CMV™-6a,b,c
N-terminal Met-FLAG (MCS in all three reading frames)
E7533, p3xFLAG-CMV™-7.1
N-terminal Met-3xFLAG
E9908, p3xFLAG-CMV™-8
N-terminal 3xFLAG (secreted)
E8658, pFLAG-Myc-CMV™-19
N-terminal FLAG, C-terminal c-Myc (Dual tagged, secreted)
E8783, pFLAG-Myc-CMV™-20
N-terminal Met-FLAG, C-terminal c-Myc (Dual tagged)
E9158, p3xFLAG-Myc-CMV™-23
N-terminal 3xFLAG, C-terminal c-Myc (Dual tagged, secreted)
E9283, p3xFLAG-Myc-CMV™-24
N-terminal Met-3xFLAG, C-terminal c-Myc (Dual tagged)
C5864, pCMV-FLAG-MAT™-1
N-terminal Met-FLAG, C-terminal MAT (Dual tagged)
C6114, pCMV-FLAG-MAT™-2
C-terminal FLAG-MAT (Dual tagged)

CMV Vector Features
CMV promoter-based vectors from Sigma-Aldrich provide flexibility in transient or stable expression, cytoplasmic expression or secretion, and N-terminal or C-terminal tagging in various combinations of FLAG, 3xFLAG, c-myc or MAT. These fusion proteins allow easy detection, purification and analysis of recombinant protein for a wide range of applications. Dual-tagged fusions provide flexibility in detection as well as a method to screen for full-length recombinant protein.

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. CMV vectors contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and the f1 origin. 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.

3xFLAG
The 3xFLAG epitope tag is 20-200 times more sensitive than the original FLAG tag. In cases of low-level expression, 3xFLAG is ideal. 3xFLAG is only 22 amino acids and is therefore unlikely to alter protein function or block other binding sites or epitopes. Like the original FLAG tag, 3xFLAG is very hydrophilic and can be cleaved with enterokinase.

MAT Tag
The MAT tag or Metal Affinity Tag (HNHRHKH) has been created for purification of recombinant MAT fusion proteins using HIS-Select Nickel and Cobalt Affinity Gels. HIS-Select products allow for highly selective purification of histidine-tagged fusion proteins such as MAT fusions. Many of our newest vectors make use of the MAT tag, often in combination with the well-known FLAG tag. MAT tag containing vectors are offered in formats for N-terminal or C-terminal tagging.

Enterokinase Removal of FLAG Tags
The recognition sequence for enterokinase, Asp-Asp-Asp-Asp-Lys, is found at the C-terminal end of the FLAG epitope tag. Removal of FLAG is possible in all fusion proteins containing an N-terminal FLAG sequence. Dual tag fusion proteins may also be cleaved with enterokinase for removal of one or more tags, depending on the position of FLAG in the protein sequence.

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