Download Fusion, Affinity and Epitope Tags Lecture Notes Handout

Biochemistry
Lab
Fusion, Affinity, a nd Epitope Tags Handout
Lecture Notes
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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
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Can be used for detection
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If antibodies are bound to beads can be used for immuno-purification
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Pull down assays
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Immunoprecipitation
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Can be placed on either terminus and still function
Pull Down Assay
Immunoprecipitation
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Tap tap
Tap tag proteins
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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
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Bind, wash, cleave then bind second tag,
wash and elute.
Biochemistry
Lab
Fusion, Affinity, a nd Epitope Tags Handout
Lecture Notes
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Common Epitope Tags
Ha tag – Hemaggllutinin
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9 amino acids YPYDVPDYA
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Sequence found on influenza protein
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Not commonly found in most cells or within protein
C-myc
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10 amino acids EQKLISEEDL
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Found on proto-oncogen involved in breast cancer and others
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Regulates gene transcription
Flag (Sigma Aldrich) aka DDK
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8 amino acids DYKDDDDK
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Sometimes found with 2 or 3 in a row for higher affinity binding
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Hydrophilic aa sequence
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First tag to be published
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May not work on both terminus
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There are different versions of the sequence
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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
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5, 6 or up to 12 Histadines placed at the terminus of a protein
Functional group of Histadine (His) is imidazole
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Negative charged aa ligands with Ni+2 bound to beads
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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
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Eluted with free imidazole, lower pH or excess EDTA
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Smallest affinity tag
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Can be used in native or denatured conditions
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pET, pQE and many other vectors offer His tags
Biochemistry
Lab
Fusion, Affinity, a nd Epitope Tags Handout
Lecture Notes
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GST – Glutatione S-transferase
GST is a full protein sequenced to the terminus of a protein
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functions in binding glutathione for redox reactions
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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
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A smallish sized protein but a large addition
Endogenous bacterial proteins do not bind to glutathione and thus lead to higher
purification
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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
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This decreases potential yield but may help folding for some high disulfide
containing proteins
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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
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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