Chapter 3 Affinity Chromatography Flashcards
Objectives
Understand the principles of affinity chromatography.
Know the linkers (spacer arms) used in affinity chromatography.
Describe the binding and elution strategies used in affinity chromatography.
Understand the importance of affinity handles (affinity tags) and fusion proteins in biotechnology.
Describe the construction of vectors used to express fusion proteins.
Describe the examples of affinity tags including the 6xHis, maltose binding protein systems, and GST.
Understand how fusion proteins are purified by affinity chromatography.
Where does chromatography fit in between protein mixture and proteins?
Sample separation and visualization
Comparative analysis
Digestion
3 uses of chromatography
- remove contaminants that might affect 2D PAGE
- to isolate specific groups of proteins for analysis using 2D PAGE
- to remove abundant non-target proteins, thus increasing the loading of rare proteins of interest during 2D-PAGE
- immunoaffinity depletion
“unwanted” proteins are trapped
Remaining “wanted” proteins flow through and collected
- affinity chromatography
“wanted” proteins trapped and eluted later
“unwanted” proteins flow through and discarded
3 principles of affinity chromatography
Based on bio-specific interaction/binding between a ligand and its target:
E.g. Antibody-antigen, Ligand-receptor, Substrate-enzyme
The binding can be ionic, hydrophobic or a mix of both.
One member is bound to a solid surface/matrix i.e. ligand
Illustration of affinity chromatography
Protein mixture is added to a column containing a polymer bound ligand specific for the protein of interest
unwanted proteins are washed through the column, which are collected in different tubes based on timings
ligand solution are poured into the column, ligands are attached to protein of interest
Typical workflow for affinity chromatography
- equilibration
- sample application, absorption of sample and flow through the unbound material
- wash away unbound materials
- elute bound proteins
- re-equilibrium
Y axis - absorbance
X axis - Column volume (CV)
Begin sample application, change in elution buffer,
Example of affinity chromatography
antibody-antigen chromatography
Load in pH 7 buffer, protein recognized by antibody, protein not recognized by antibody flow out
wash
elute with pH 3 buffer into different tubes containing the proteins
2 Purpose of equilibration
To ensure that the buffer conditions in the column is equivalent to that of the protein sample.
Prevents accidental denaturation, precipitation etc.
2 purpose of regeneration
If the column matrix is to be re-used, it needs to be washed with buffers containing denaturants, chaotropes etc. to remove residual bound proteins.
The column can then be re-equilibrated for future use.
The 3 principles of affinity chromatography
Ligand is immobilized to stationary matrix through a spacer arm
Target molecules bind to ligand and stay in column
Target molecules eluted by a change in conditions e.g change in pH
2 purpose of regeneration
If the column matrix is to be re-used, it needs to be washed with buffers containing denaturants, chaotropes etc. to remove residual bound proteins.
The column can then be re-equilibrated for future use
Spacer arm
the chain of carbon and/or other atoms that positions a functional group away from the solid matrix to which it is covalently bound and makes it more available to a ligand and less restricted by steric hindrance by the matrix.
illustration of affinity chromatography
matrix is bound to spacer arm, spacer arm is bound to ligand, target is bound to ligand
3 characteristics of the matrix
- physically stable to withstand high pressure in column
- chemically stable to withstand harsh environments during operation of the column
- allow derivatization for linking of spacer or ligand
Sepharose
Beaded agarose is typically used as a matrix
Describe sepharose
Consists of cross-linked agarose that resists denaturation.
Characteristics of ligands
- Bind with a high specificity to target proteins and easily coupled to the matrix
- form a stable yet reversible complex with target proteins
- easy to dissociate target proteins without adverse biochemical changes
What ligands are
- small molecules (glutathione) or macromolecular (protein A, antibodies, lectins)
2 types of ligand
Mono specific or group specific
Examples of mono specific ligands
Anti-Bovine Serum Albumin mAb
Example of group specific ligands
Protein A, lectins
A common problem that can arise with affinity chromatography
Inefficient target protein binding and subsequent recovery
Why inefficient target protein binding and subsequently recovery?
Due to stearic hindrance between target proteins especially if ligand is much smaller relative to the target protein
Characteristics of spacer arm
The use of a spacer arm or linker to extend ligand away from matrix.
Consists of an inert hydrocarbon chain structure.
However, long spacer arms can create nonspecific hydrophobic interactions
Illustration of spacer arm
overlap between target molecules and the bead surface may prevent binding between ligand and target molecule
spacer arm ensures that the target molecule has full access to ligand
Ligand matrix coupling
Using the cyanogen bromide activation
Cyanogen bromide (CNBr) activation
CNBr activation couples amino-group containing ligands such as proteins and antibodies to Sepharose.
3 steps in cyanogen bromide
1 - activation of hydroxyl groups by cyanogen bromide
2 - forming of reactive cyanate ester
3 - covalent binding of ligands via amino groups
Choice of elution method
Disruption of ligand-protein interaction results in elution of target proteins
affinity elution is usually on the form of a gradient
4 disruptions of ligand-protein interaction that results in elution of target proteins
Change in pH
Change in ionic strength
Use of a denaturant
Use of competitor (e.g. free reduced glutathione to displace GST-bound fusion proteins)
affinity elution is usually in the form of a gradient
3 choices of elution methods
- step elution
- gradient elution
- isocratic elution
3 choices of elution conditions
step elution - stepwise change
gradient elution - linear change
isocratic elution - no change in conditions
Gradual change (gradient) vs no change (isocratic)
choice of elution method: gradient elution
characteristics of gradient elution
Weakly bound proteins are first eluted
Compression of peak
Better separation and resolution, and complete elution
Gradient elution
Linear change in elution conditions and used to identify optimal elution of target protein
- Start of selected imidazole concentration for elution of (His)6 fusion protein
- End of selected imidazole concentration for elution of (His)6 fusion protein
programmed elution buffer concentration
(His)6, fusion protein
types of ligand-target combination
Streptavidin-biotin
Streptavidin-biotin
- this Streptavidin-biotin bond is one of the strongest non-covalent interactions known
- proteins can be biotinylated and purified by streptavidin-Sepharose.
Streptavidin
Comes from streptomyces avidinii
What do the black spots represent
Membrane proteins of H. pylori biotinylated and purified with avidin-Sepharose
Proteins visualised on 2D-gel by HRP-conjugated avidin
types of ligand target combinations
Protein A/G-immunoglobulins (Ig)
Protein A/G-immunoglobulins (Ig)
Fusion protein that comprises IgG binding domains from Protein A and G.
Binds to all human Ig subclasses at varying extents.
Binds to all subclasses of mouse IgG but does not bind mouse IgA, IgM or serum albumin.
Useful for purifying mouse IgG mAbs.
Protein A
Surface protein in the cell wall of Staphylococcus aureus
Has the ability to bind immunoglobulins (Ig)
Protein G
Expressed by Streptococcal bacteria
Also binds to immunoglobulins (Ig)
Difference between protein A and G
protein A and G have different binding specificities
Types of ligand-target combinations
Lectins-glycans
Lectins-glycans
Lectins are plant-derived macromolecules that bind with a high specificity to sugars.
(e.g. concanavalin A, phytohemagglutinin, ricin)
Used extensively to purify glycoproteins.
Lectins can be used to differentiate terminal sugars on glycans i.e. glycoproteome chip
Lectins Concanavalin A
Source Jackbean seeds
Sugar specificity - alpha D mannose, alpha D glucose
Eluting sugar - alpha D methylmannose
Lectins wheat germ agglutin
Source wheat germ
Sugar specificity - N-acetyl-beta-D-glucosamine, N acetyl-beta-D neuraminic acid
Eluting sugar - N-acetyl-beta-D-glucosamine
Lectins Pisum sativum lectin (PSA)
Source peas
Sugar specificity alpha D mannose
Eluting sugar alpha D methylmannose
Lectins soybean lectin (SBA)
Source Soybean
Sugar specificity N-acetyl-beta-D-galactosamine
Eluting sugar N-acetyl-beta-D-galactosamine
Affinity purification of target proteins
Epitope tags can be added to recombinant proteins for the purpose of affinity purification (affinity tags).
N terminal (5’ end) or C terminal (3’ end) tag fused to a target protein = fusion protein e.g. HA, FLAG, c-myc, GST, 6xHis
Tag can be enzymatically cleaved off (e.g. thrombin) to facilitate recovery of native protein
Epitope Tag
Epitope tagging is a technique in which a known epitope is fused to a recombinant protein by means of genetic engineering. By choosing an epitope for which an antibody is available, the technique makes it possible to detect proteins for which no antibody is available. This is especially useful for the characterization of newly discovered proteins and proteins of low immunogenicity.
HA Tag
(= haemagglutinin tag) short polypeptide (YPYDVPDYA) derived from the influenza virus haemagglutinin molecule.
FLAG-tag, or FLAG octapeptide
The short synthetic peptide sequence of the FLAG-tag (DYKDDDDK) can be removed via cleavage by enterokinase.
Glutathione S-transferase (GST)
a 26kDa protein which can is used in GST-pull down assays to purify GST-fused recombinant proteins.
6xHis-tag
an oligohistidine domain that will bind to a matrix containing nickel ions (Ni2+) chelated to nitriloacetic acid-agarose.
Affinity tags serve 2 other functions besides allowing highly specific purification
Solubilization tags
Used for visualization via staining or western blot
Solubilization tags
Are used for recombinant proteins expressed in chaperone-deficient species (e.g. E. coli) to prevent protein aggregation.
Used for visualization via staining or western blot
Used for visualisation via staining or Western blot. Enzyme-linked Anti-HA and Anti-FLAG antibodies are useful for visualization by Western blots or ELISA
Affinity chromatography
GST tag system
in which the functional GST protein (26 kDa) is fused to the N-terminus of the recombinant protein.
GST is a 211 amino acid protein (26 kDa) whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins.
GST folds rapidly into a stable and highly soluble protein upon translation, inclusion of the GST tag often promotes greater expression and solubility of recombinant proteins than expression without the tag.
In addition, GST-tagged fusion proteins can be purified or detected based on the ability of GST (an enzyme) to bind its substrate, glutathione
6xHis or poly-histidine tag system
Flow-through run on SDS-PAGE
Lane 1: starting material
Lane 2: column flow-through
Lane 3: wash 1
Lane 4: elution
illustration of 6xHis Tag system
matrix - spacer arm - nitrilotriacetic acid and nickel form oligohistidine domain - binds to multiple proteins
6xhis tag system
Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli [4] and other prokaryotic expression systems. Bacterial cells are harvested via centrifugation and the resulting cell pellet lysed either by physical means or by means of detergents and enzymes such as lysozyme or any combination of these. At this stage, raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with an affinity resin containing bound divalent nickel or cobalt ions, which are available commercially in different varieties. Nickel and cobalt have similar properties and as they are adjacent period 4 transition metals (v. iron triad). These resins are generally sepharose/agarose functionalized with a chelator, such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl-methyl aspartate (Co-CMA) for cobalt, which the polyhistidine-tag binds with micromolar affinity. Ernst Hochuli et al. coupled 1987 the NTA ligand and Nickel-ions to agarose beads.[5] The resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion. With Ni-based methods, washing efficiency can be improved by the addition of 20 mM imidazole (proteins are usually eluted with 150-300 mM imidazole). Generally, nickel-based resins have a higher binding capacity, while cobalt-based resins offer the highest purity. The purity and amount of protein can be assessed by SDS-PAGE and Western blotting.[citation needed]
Affinity purification using a polyhistidine-tag usually results in relatively pure protein when the recombinant protein is expressed in prokaryotic organisms. Depending on downstream applications, including the purification of protein complexes to study protein interactions, purification from higher organisms such as yeasts or other eukaryotes may require a tandem affinity purification[6] using two tags to yield higher purity. Alternatively, single-step purification using immobilized cobalt ions rather than nickel ions generally yields a substantial increase in purity and requires lower imidazole concentrations for elution of the his-tagged protein.
Polyhistidine-tagging is the option of choice for purifying recombinant proteins in denaturing conditions because its mode of action is dependent only on the primary structure of proteins. For example, even when a recombinant protein forcibly expressed in E. coli produces an inclusion body and can not be obtained as a soluble protein, it can be purified with denaturation with urea or guanidine hydrochloride. Generally, for this sort of a technique, histidine binding is titrated using pH instead of imidazole binding—at a high pH, histidine binds to nickel or cobalt, but at low pH (~6 for cobalt and ~4 for nickel), histidine becomes protonated and is competed off of the metal ion. Compare this to antibody purification and GST purification, a prerequisite to which is the proper (native) folding of proteins involved. On the other hand, it is said that the His tag tends to aggregate and insolubilize more than other affinity tags.
Polyhistidine-tag columns retain several well known proteins as impurities. One of them is FKBP-type peptidyl prolyl isomerase, which appears around 25kDa (SlyD). Impurities are generally eliminated using a secondary chromatographic technique, or by expressing the recombinant protein in a SlyD-deficient E. coli strain.[7] Alternatively, comparing with nickel-based, cobalt-based resins have less affinity with SlyD from E. coli, but in several cases, it is moderately helpful.[8]
Affinity chromatography
Maltose-binding protein (MBP) tag
MBP is a part of the maltose/maltodextrin metabolism in E. coli, responsible for the uptake and efficient catabolism of maltodextrins.
MBP is expressed as a tag to bind to amylose-Sepharose beads.
MBP improves solubility of the fusion protein.
Eluted with maltose/amylose as competitor or cleaved.
Steps involved in amylose affinity chromatography
① Clone and express the target protein as a maltose binding protein (MBP) fusion protein, with an engineered proteolytic cleavage site.
② Apply the MBP fusion protein to the amylose affinity column.
③ The MBP fusion protein binds to the amylose affinity column.
④ Elute the MBP fusion protein using maltose/amylose.
⑤ Cleave the MBP fusion protein with a specific protease to release the target protein from MBP.
