Molecular Cloning & Protein Expression

CMV promoter mammalian protein expression vector

The aim of molecular cloning is to insert the gene-of-interest (GOI) into a plasmid vector, a circular piece of DNA that contains various elements to facilitate cloning, clone selection, and protein expression. Researchers often use DNA restriction enzymes and ligase to insert the GOI in-frame within the expression vector for subsequent protein expression. This vector is then inserted into a cell that will express the protein encoded by the GOI or protein expression is accomplished using cell-free protein synthesis systems.

Once the protein is expressed, the protein function can be studied as it affects the cell signaling, morphology, or other aspects within the cell. Alternatively, the protein can be expressed in large quantities, purified, and used in numerous downstream applications. For additional information, explore our comprehensive offering of reliable reagents and resources that are available across the cloning & expression workflow for insect, bacteria, and mammalian protein expression systems.

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Mammalian Expression Vectors Using CMV Promoters and FLAG® Tag Vectors

What is a CMV promoter? Human cytomegalovirus (CMV) promoter is an expression vector used to drive gene expression. A strong promoter is one of two elements required for active gene expression. It is a region of DNA that controls transcription of a particular gene, which ensures that the expression vector is able to produce enough protein for further downstream assays and analyses.

CMV, FLAG®, and 3xFLAG® Tag Features

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. The 3xFLAG® epitope tag is 20-200 times more sensitive than the original FLAG® tag. In cases of low-level expression, the 3xFLAG® epitope is ideal. The 3xFLAG® sequence 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, the 3xFLAG® sequence is very hydrophilic and can be cleaved with enterokinase. Discover more about FLAG® tag technology and browse our complete list of FLAG® and 3xFLAG® vectors, as well as our Anti-FLAG® antibody products.

Insect Cell Protein Expression System

Baculovirus-mediated expression of recombinant proteins in insect cells is a valuable tool for producing soluble, active, individual or multimeric protein complexes, virus-like particles, and membrane proteins. BacMagic™ DNA kits save time by generating recombinant baculoviruses for target protein expression in insect cells without the tedious, time-consuming

plaque purification steps. The BacMagic™ System improves on the traditional method for generating recombinant baculoviruses by eliminating time-consuming plaque purification. For additional information, please explore our comprehensive offering of molecular cloning and protein expression resources to find the best reagent or protocol for your specific application need.

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