Term
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Definition
Affinity Chromatography Binding to Another Molecule
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Term
| Affinity Chromatography (AC) |
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Definition
Used to purify specific biological macromolecules
Proteins
Nucleic acids
Stationary phase – immobilized molecule that binds with high specificity to a single protein
Most molecules wash away
A few macromolecules interact with high specificity with the immobilized molecule and are retained on the stationary phase |
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Term
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Definition
The specificity of these physiologically relevant interactions derives from complementary surfaces on the interacting molecules
Often involves several different types of interactions, such as hydrogen bonding and hydrophobic interactions
The selectivity of affinity chromatography is potentially the highest of any form of chromatography |
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Term
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Definition
Approach: chemically couple a small molecule or protein to an inert material
Dextran, polyacrylamide, or agarose provides a stable open gel structure, which large proteins can penetrate, and a large surface area to which the immobilized group (affinity ligand) can be linked
These stationary matrixes can be formed into beads with a large number of reactive groups
Affinity ligand is commonly linked to free hydroxyl groups on the matrix |
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Term
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Definition
After washing the noninteracting molecules away, the target molecules are eluted with specific substances
Highly specific elution of the desired macromolecule from the stationary phase is achieved by adding a gradient of a competing agent to the buffer, which is usually the same molecule that was immobilized on the column
Used to purify specific biological macromolecules:
Enzyme bound to substrate Mobilized substrate
GST/GSH
MBP/Maltose
Antibody bound to antigen Mobilized antigen
Hormone bound to receptor Mobilized receptor
Metal bound to histidines Histidine ring |
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Term
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Definition
Gene Fusion
GST column
Glutathione binds glutathione-S-transferase
Elute with glutathione
MBP column
Amylose binds MBP
Elute with maltose
Metal chelate chromatography
Nickel column
Nickel (and other metals such as cobalt) bind 6-8 histadines
Elute with imidazole |
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Term
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Definition
The gene encoding the protein of interest is fused to a second gene encoding a protein that is easily purified by affinity chromatography
This second protein is called the “tag”
The vector that one uses for cloning is a plasmid containing the tag sequence
Constructed with specific restriction sites that permit fusing the gene of interest to the tag gene in the same reading frame
This gene fusion encodes a large protein consisting of the protein of interest fused to the tag protein |
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Term
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Definition
Types
Glutathione-S-transferase (GST)
Maltose-binding protein (MBP)
Calmodulin-binding protein (CBP)
Streptavidin
FLAG
Thioredoxin (Trx)
NusA protein (Nus)
DsbA, DsbC
Green fluorescent protein (GFP)
C2 N-terminus (botulism toxin)
Chitin binding
Can allow for enzymatic tracking (GST CDNB Assay)
Can enhance solubility (GST, Trx, MBP, Nus)
Can provide an enzyme to catalyze disulfide bonds (Trx)
Provide signal sequence folding and export (Dsb), or translocation into mammalian cells (botulism toxin)
Tracking a protein through a cell (GFP) |
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Term
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Definition
| Often the fusion vector is designed to introduce a short “linker” amino acid sequence between the protein of interest and the tag protein (MBP) |
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Term
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Definition
This linker region can contain amino acid sequences that are cleavage sites for particular proteases
The plasmid vector can be designed to have a strong promoter that provides a high level of transcription of the fusion gene upon induction |
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Term
| Expression and Purification Strategy |
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Definition
Construct fusion gene and introduce into bacteria
Grow bacteria, express the fusion gene, harvest cells and make crude extract
Run crude extract on amylose AC column to purify the fusion protein
The purified fusion protein is cleaved with Factor Xa protease (thrombokinase), separating the target protein from maltose-binding protein (MBP)
Rerun sample on the amylose column to separate the target protein from MBP |
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Term
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Definition
Glutathione-s-transferase (GST) binds glutathione on beads
Elute with glutathione (GSH) |
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Term
| Green Fluorescent Protein (GFP) |
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Definition
| Not for purification, but tracking |
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Term
| Metal-Chelate Chromatography |
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Definition
Developed in early 1960s
IMAC – immobilized metal affinity chromatography
A type of affinity chromatography based on the chelation of metals such as nickel or cobalt
Approach takes advantage of the fact that stretches of histidine residues bind tightly to metals such as nickel
The gene of interest is genetically manipulated to encode a polyhistidine tag, usually six to eight histidines in length, at either the N- or C-terminus of the protein |
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Term
| Metal-Chelate Chromatography |
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Definition
The presence of a few extra histidine residues at the end of the protein does not usually affect the activity of the protein
The fusion protein with the histidine tag is then passed through a column containing immobilized nickel to which it adheres
Then the fusion protein is specifically eluted from this column by imidazole, which competes with histidine for the immobilized nickel |
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Term
| Tandem Affinity Purification |
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Definition
To purify proteins with the TAP protocol, apply the mammalian cell lysate to the streptavidin resin, elute using biotin, and apply that eluate to a calmodulin resin.
Once you elute with EGTA, you will get exceptionally pure proteins. |
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Term
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Definition
Many salts increase the level of intermolecular structures in water
Addition of these salts increases the strength of hydrophobic interactions (salting-out)
Often this can be an excellent purification step, particularly when the protein of interest differs significantly in solubility from most other proteins in the crude extract
Ammonium sulfate
Used to clarify crude extracts
Stabilizes proteins
Gentle method for purification |
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Term
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Definition
Salts that reduce the amount of structure (chaotropic) by breaking weak bonds
6M Guanidine-HCl, urea
Useful for protein refolding of inclusion bodies |
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Term
| Hydrophobic Interaction Chromatography (HIC) |
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Definition
Proteins contain a variety of amino acid residues, some with polar and nonpolar (hydrophobic) side chains
Many of the hydrophobic side chains are buried in the interior of the proteins, away from the surrounding water
Exposed surfaces of proteins are rich in amino acids with polar side chains
Some proteins have hydrophobic amino acids on their surface
Protein-protein interactions
Protein-lipid membrane interactions
The differences in the hydrophobicity of the surfaces of proteins are exploited by hydrophobic interaction chromatography
Aggregation of solute molecules is thermodynamically favorable |
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Term
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Definition
The stationary phase is usually an inert material with hydrophobic functional groups coupled to it
ethyl, propyl, butyl, hexyl, octyl, and phenyl
Method:
Apply sample in a buffer that is highly polar (high salt concentration such as ammonium sulfate, which enhances hydrophobic interactions) to column
After non-binding proteins have been washed off the column, bound proteins are eluted by reducing the salt concentration
(polar nonpolar)
The least hydrophobic proteins elute first, and more hydrophobic proteins are released at lower concentrations of salt |
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Term
| Reverse-Phase Chromatography |
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Definition
Type of HIC for small molecules
Peptide purification
Long-chain hydrocarbon (C8-C18) linked to an inert silica-based support
Performed under denaturing conditions – separation based on hydrophobicity
Elution of peptides with acetonitrile or methanol in the order of their increasing hydrophobicity |
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Term
| pH and Differential Solubility |
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Definition
Proteins are least soluble and often precipitate when the pH of the solution equals their pI, when the net charge on the protein is zero
This property may be used to selectively precipitate the protein of interest or to precipitate contaminants
Most useful when pIs are different |
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Term
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Definition
Alcohols, acetone
Denature, inactivate many proteins
Large volumes of organic solvents not preferred
However, for proteins that survive the treatment, this can be a powerful fractionation step and has the additional advantage of helping to clarify the extract |
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