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Download Fusion, Affinity and Epitope Tags Lecture Notes Handout
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Biochemistry Lab Fusion, Affinity, a nd Epitope Tags Handout Lecture Notes Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Tags – What are they? Affinity and Epitope tags are additional amino acids added to the carboxy or amino – terminal of a recombinant protein Fusion proteins (also called chimeric proteins) are full sized proteins genetically coded on a recombinant protein For purification (affinity) Finding binding proteins (proteomics) Determining cellular location Assessing expression Detect movement within cells Enhance yield and solubility of proteins A little more specific... Epitope tag – a short sequence of amino acids recognized by antibodies (antigenic determinant) IMPORTANT POINT – they are small amino acid peptides (8 – 15 aa total) which do not add a significant size or structural barrier to protein function ü Can be used for detection ü If antibodies are bound to beads can be used for immuno-purification ü Pull down assays ü Immunoprecipitation ü Can be placed on either terminus and still function Pull Down Assay Immunoprecipitation Ø Tap tap Tap tag proteins ü Tandam Affinity Purification (TAP) • Two epitope tags on protein (typically on same terminus) • Used to isolate low abundant protein for Mass Spectroscopy Analysis – can determine protein captured or proteins which bind to the tagged protein ü Bind, wash, cleave then bind second tag, wash and elute. Biochemistry Lab Fusion, Affinity, a nd Epitope Tags Handout Lecture Notes Ø Ø Ø Ø Ø Ø Common Epitope Tags Ha tag – Hemaggllutinin ü 9 amino acids YPYDVPDYA ü Sequence found on influenza protein ü Not commonly found in most cells or within protein C-myc ü 10 amino acids EQKLISEEDL ü Found on proto-oncogen involved in breast cancer and others ü Regulates gene transcription Flag (Sigma Aldrich) aka DDK ü 8 amino acids DYKDDDDK ü Sometimes found with 2 or 3 in a row for higher affinity binding ü Hydrophilic aa sequence ü First tag to be published ü May not work on both terminus ü There are different versions of the sequence ü Some antibodies (M1, M2, M5) only recognize one version! Affinity Tags Small protein, large regions (domains) of a protein or whole protein genetically sequenced to the N or C terminus of a protein Used for affinity purification BUT can also be used as epitope tags His tag is often placed as an epitope tag but shouldn’t be as its key function is for purification His Tag Ø 5, 6 or up to 12 Histadines placed at the terminus of a protein Functional group of Histadine (His) is imidazole ü Negative charged aa ligands with Ni+2 bound to beads ü Binding may improve with amino acid spacers – His-x-x-His-x-x this aligns imidazole ring to same side and allows for flexibility to wrap around Ni+2 Ø Eluted with free imidazole, lower pH or excess EDTA Ø Smallest affinity tag Ø Can be used in native or denatured conditions Ø pET, pQE and many other vectors offer His tags Biochemistry Lab Fusion, Affinity, a nd Epitope Tags Handout Lecture Notes Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø GST – Glutatione S-transferase GST is a full protein sequenced to the terminus of a protein ü functions in binding glutathione for redox reactions ü GST can bind to beads covalently bound to glutathione ü Easy removal from beads by adding free glutathione or protease of protein sequence between two proteins GST is a modestly large – 26 kDa ü A smallish sized protein but a large addition Endogenous bacterial proteins do not bind to glutathione and thus lead to higher purification ü The same can not be said for His Is immunogenic – must remove before using recombinant protein for antibody production (not His) Main vector is pGEX Maltose Binding Protein (MBP) Large protein which binds to amylose Secreted into periplamsic space of E. coli where disulfide bonds can more favorably form ü This decreases potential yield but may help folding for some high disulfide containing proteins th 1/5 of MBP fusion proteins do not bind to affinity resin MBP can help expression by increasing solubility more than others (avoids inclusion bodies). Fusion Protein Similar to epitope or affinity tags but the main function is NOT for antibody use (epitope tag) or for purification (affinity tag) Term often used interchangably with all three – but shouldn’t be Generated by cloning of two genes with the removal of stop codon of first gene Key examples include GFP, and other color variants Fusion Protein Aids with solubility or expression ü Second full protein can mask some issues Often used for detection in cells – translocation in living cells (GFP) Improved detection with better antibodies Can be a problem with size and often need controls to show there is a minimized effect