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7.25F: Purifying Proteins by Affinity Tag

  • Page ID
    9504
  • [ "article:topic", "authorname:boundless", "Affinity Tags" ]

    Protein tags are peptide sequences genetically grafted onto a recombinant protein.

     

    LEARNING OBJECTIVES

    Indicate the uses of protein affinity tags

     

    KEY TAKEAWAYS

    Key Points

    • Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique.
    • Recombinant proteins that carry small affinity tags are efficiently expressed in bacteria, insect, or mammalian cells.
    • After cell lysis and clearing of the lysate, tagged proteins are purified using an immobilized-metal affinity chromatography procedure.

     

    Key Terms

    • protein: Proteins are large biological molecules consisting of one or more chains of amino acids.
    • affinity: An attractive force between atoms, or groups of atoms, that contributes toward their forming bonds.
    • recombinant: This term refers to something formed by combining existing elements in a new combination. Thus, the phrase recombinant DNA refers to an organism created in the lab by adding DNA from another species.

    Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes.

    Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique. These include chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST). The poly (His) tag is a widely-used protein tag; it binds to metal matrices.

    image

    Adding Polyhistidine Tags: This is an example of a primer designed to add a 6xHis-tag using PCR. Eighteen bases coding six histidines are inserted right after the START codon or right before the STOP codon. At least 16 bases specific to the gene of interest are needed next to the His-tag. With 6 His, the protein will have an added 1 kDa of molecular weight. Oftentimes, a linker (such as gly-gly-gly or gly-ser-gly) is placed between the protein of interest and the 6 His tag. This is to prevent the polyhistidine tag from affecting the activity of the protein being tagged.

    Solubilization tags are used, especially for recombinant proteins expressed in chaperone-deficient species such as E. coli, so as to assist in the proper folding in proteins and keep them from precipitating. These include thioredoxin (TRX) and poly (NANP). Some affinity tags have a dual role as a solubilization agent, such as MBP and GST.

    Chromatography tags are used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique. These often consist of polyanionic amino acids, such as FLAG-tag.

    Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include V5-tag, c-myc-tag, and HA-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification.

    Fluorescence tags are used to give visual readout on a protein. GFP and its variants are the most commonly used fluorescence tags. More advanced applications of GFP include using it as a folding reporter (fluorescent if folded, colorless if not).

    Protein tags are also useful for specific enzymatic modification (such as biotin ligase tags) and chemical modification (FlAsH) tag. Often tags are combined to produce multifunctional modifications of the protein. However, with the addition of each tag comes the risk that the native function of the protein may be abolished or compromised by interactions with the tag.

    Examples of peptide tags include:

    • AviTag, a peptide allowing biotinylation by the enzyme BirA and so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE)
    • Calmodulin-tag, a peptide bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL)
    • FLAG-tag, a peptide recognized by an antibody (DYKDDDDK)
    • HA-tag, a peptide recognized by an antibody (YPYDVPDYA)
    • His-tag, 5-10 histidines bound by a nickel or cobalt chelate (HHHHHH)
    • Myc-tag, a short peptide recognized by an antibody (EQKLISEEDL)
    • S-tag (KETAAAKFERQHMDS)
    • SBP-tag, a peptide which binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)
    • Softag 1, for mammalian expression (SLAELLNAGLGGS)
    • Softag 3, for prokaryotic expression (TQDPSRVG)
    • V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST)
    • Xpress tag (DLYDDDDK)

    Examples of protein tags include:

    • BCCP (Biotin Carboxyl Carrier Protein), a protein domain recognized by streptavidin
    • Glutathione-S-transferase-tag, a protein which binds to immobilized glutathione
    • Green fluorescent protein-tag, a protein which is spontaneously fluorescent and can be bound by nanobodies
    • Maltose binding protein-tag, a protein which binds to amylose agarose
    • Nus-tag
    • Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK)
    • Thioredoxin-tag