During the construction of expression vectors, we usually add different tags to the vectors aim to simplify downstream detection and purification. Series of pcDNA3.1 vectors, as the widely used mammalian expression vectors, which can be added to many types of protein tags. The tags can be fused to the N-terminus or C-terminus of the target protein to meet the demand for protein expression, detection and purification of the target protein. Depending on the sizes of the molecular weight of the tags, the tags can usually be divided into two categories, one is a short peptide tag that binds to an immobilized ligand, such as His-tag, Flag-tag, etc.; the other is a protein tag that recognizes a small molecule ligand, such as GST, MBP, etc., which are used in ELISA, Western blot (WB), Flow Cytometry, Immunoprecipitation (IP) and Immunofluorescence (IF).
The tags of pcDNA3.1 vectors
l DYK tag: A short peptide composed of 8 amino acids (DYKDDDDK), which can be fused to the N-terminus or C-terminus of the target protein and used for the immunoadsorption purification of recombinant protein. The molecular weight of the DYK tag is small and it will not affect the biological activity of the target protein.
l His tag: A short peptide composed of 6 histidines (His-His-His-His-His-His) which can be fused to the N-terminus or C-terminus of the target protein. Due to its small molecular weight, it is easier to separate and purify and can be used for Adsorption and purification of recombinant protein. His tag is currently the most widely used tag.
l HA tag: A short peptide composed of 9 amino acids (YPYDVPDYA), which can be fused to the N-terminus or C-terminus of the target protein. Because of its small molecular weight, it has little effect on the target protein and is widely used for the separation, purification, detection, and tracing of HA fusion target protein.
l C-Myc: A short peptide composed of 11 amino acids (EQKLISEEDL), which can be fused to the N-terminus or C-terminus of the target protein. Due to the small molecular weight, there is no interference with the fused protein. It is widely used for detection but rarely used for purification. C-Myc tag has been successfully used in Western-blot hybridization technology, immunoprecipitation and flow cytometry. It can be used to detect the expression of recombinant protein in target cells.
l GST tag: The molecular weight is relatively large, about 26KD, which can be fused to the C-terminus or N-terminus of the target protein. GST fusion protein is soluble, so it is often used to increase protein solubility and expression. After the fusion protein is expressed, you can consider whether to remove the GST tag according to the downstream application, to avoid the immunological impact of the protein tag on the target protein. If you want to remove the GST tag protein, you can use site-specific protease excision.
l eGFP tag: Enhanced green fluorescent protein, which can be used as a reporter gene to construct into a target vector, express fusion protein to detect the location, migration, conformational change and interaction between molecules of target protein molecules, or to target certain organelles. eGFP can be expressed in bacteria, yeast, plants and mammals and produces visible fluorescence and it has stable fluorescence properties.
l TEV/ Thrombin: A protease that is widely used to cleave tags. You can use the TEV enzyme or Thrombin enzyme to separate the GST tag from the target protein.
l P2A: Use the linker, P2A, to drive the expression of multiple genes. P2A can be used to separate each gene, and a single mRNA transcript will produce multiple proteins.
l Hygro/Zeo: Resistance tags for hygromycin selection and bleomycin selection.
GenScript provides a series of pcDNA3.1 vectors with a variety of labels to meet your experimental applications. Learn more detailed information：www.genscript.com/gene-cloning-subcloning.html