Chapter 3 Affinity Chromatography

Question 1

Objectives

Answer 1

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.

Question 2

Where does chromatography fit in between protein mixture and proteins?

Answer 2

Sample separation and visualization
Comparative analysis
Digestion

Question 3

3 uses of chromatography

Answer 3

• 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

Question 4

1. immunoaffinity depletion

Answer 4

“unwanted” proteins are trapped

Remaining “wanted” proteins flow through and collected

Question 5

2. affinity chromatography

Answer 5

“wanted” proteins trapped and eluted later

“unwanted” proteins flow through and discarded

Question 6

3 principles of affinity chromatography

Answer 6

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

Question 7

Illustration of affinity chromatography

Answer 7

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

Question 8

Typical workflow for affinity chromatography

Answer 8

1. equilibration
2. sample application, absorption of sample and flow through the unbound material
3. wash away unbound materials
4. elute bound proteins
5. re-equilibrium

Y axis - absorbance
X axis - Column volume (CV)

Begin sample application, change in elution buffer,

Question 9

Example of affinity chromatography

Answer 9

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

Question 10

2 Purpose of equilibration

Answer 10

To ensure that the buffer conditions in the column is equivalent to that of the protein sample.

Prevents accidental denaturation, precipitation etc.

Question 11

2 purpose of regeneration

Answer 11

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.

Question 12

The 3 principles of affinity chromatography

Answer 12

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

Question 13

2 purpose of regeneration

Answer 13

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

Question 14

Spacer arm

Answer 14

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.

Question 15

illustration of affinity chromatography

Answer 15

matrix is bound to spacer arm, spacer arm is bound to ligand, target is bound to ligand

Question 16

3 characteristics of the matrix

Answer 16

• 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

Question 17

Sepharose

Answer 17

Beaded agarose is typically used as a matrix

Question 18

Describe sepharose

Answer 18

Consists of cross-linked agarose that resists denaturation.

Question 19

Characteristics of ligands

Answer 19

• 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

Question 20

What ligands are

Answer 20

• small molecules (glutathione) or macromolecular (protein A, antibodies, lectins)

Question 21

2 types of ligand

Answer 21

Mono specific or group specific

Question 22

Examples of mono specific ligands

Answer 22

Anti-Bovine Serum Albumin mAb

Question 23

Example of group specific ligands

Answer 23

Protein A, lectins

Question 24

A common problem that can arise with affinity chromatography

Answer 24

Inefficient target protein binding and subsequent recovery

Question 25

Why inefficient target protein binding and subsequently recovery?

Answer 25

Due to stearic hindrance between target proteins especially if ligand is much smaller relative to the target protein

Question 26

Characteristics of spacer arm

Answer 26

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

Question 27

Illustration of spacer arm

Answer 27

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

Question 28

Ligand matrix coupling

Answer 28

Using the cyanogen bromide activation

Question 29

Cyanogen bromide (CNBr) activation

Answer 29

CNBr activation couples amino-group containing ligands such as proteins and antibodies to Sepharose.

Question 30

3 steps in cyanogen bromide

Answer 30

1 - activation of hydroxyl groups by cyanogen bromide

2 - forming of reactive cyanate ester

3 - covalent binding of ligands via amino groups

Question 31

Choice of elution method

Answer 31

Disruption of ligand-protein interaction results in elution of target proteins

affinity elution is usually on the form of a gradient

Question 32

4 disruptions of ligand-protein interaction that results in elution of target proteins

Answer 32

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

Question 33

3 choices of elution methods

Answer 33

• step elution
• gradient elution
• isocratic elution

Question 34

3 choices of elution conditions

Answer 34

step elution - stepwise change

gradient elution - linear change

isocratic elution - no change in conditions

Question 35

Gradual change (gradient) vs no change (isocratic)

choice of elution method: gradient elution

characteristics of gradient elution

Answer 35

Weakly bound proteins are first eluted

Compression of peak

Better separation and resolution, and complete elution

Question 36

Gradient elution

Answer 36

Linear change in elution conditions and used to identify optimal elution of target protein

Question 37

1. Start of selected imidazole concentration for elution of (His)6 fusion protein
2. End of selected imidazole concentration for elution of (His)6 fusion protein

Answer 37

programmed elution buffer concentration

(His)6, fusion protein

Question 38

types of ligand-target combination

Streptavidin-biotin

Answer 38

Streptavidin-biotin

• this Streptavidin-biotin bond is one of the strongest non-covalent interactions known
• proteins can be biotinylated and purified by streptavidin-Sepharose.

Question 39

Streptavidin

Answer 39

Comes from streptomyces avidinii

Question 40

What do the black spots represent

Answer 40

Membrane proteins of H. pylori biotinylated and purified with avidin-Sepharose

Proteins visualised on 2D-gel by HRP-conjugated avidin

Question 41

types of ligand target combinations

Answer 41

Protein A/G-immunoglobulins (Ig)

Question 42

Protein A/G-immunoglobulins (Ig)

Answer 42

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.

Question 43

Protein A

Answer 43

Surface protein in the cell wall of Staphylococcus aureus

Has the ability to bind immunoglobulins (Ig)

Question 44

Protein G

Answer 44

Expressed by Streptococcal bacteria

Also binds to immunoglobulins (Ig)

Question 45

Difference between protein A and G

Answer 45

protein A and G have different binding specificities

Question 46

Types of ligand-target combinations

Lectins-glycans

Answer 46

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

Question 47

Lectins Concanavalin A

Answer 47

Source Jackbean seeds
Sugar specificity - alpha D mannose, alpha D glucose
Eluting sugar - alpha D methylmannose

Question 48

Lectins wheat germ agglutin

Answer 48

Source wheat germ
Sugar specificity - N-acetyl-beta-D-glucosamine, N acetyl-beta-D neuraminic acid
Eluting sugar - N-acetyl-beta-D-glucosamine

Question 49

Lectins Pisum sativum lectin (PSA)

Answer 49

Source peas
Sugar specificity alpha D mannose
Eluting sugar alpha D methylmannose

Question 50

Lectins soybean lectin (SBA)

Answer 50

Source Soybean
Sugar specificity N-acetyl-beta-D-galactosamine
Eluting sugar N-acetyl-beta-D-galactosamine

Question 51

Affinity purification of target proteins

Answer 51

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

Question 52

Epitope Tag

Answer 52

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.

Question 53

HA Tag

Answer 53

(= haemagglutinin tag) short polypeptide (YPYDVPDYA) derived from the influenza virus haemagglutinin molecule.

Question 54

FLAG-tag, or FLAG octapeptide

Answer 54

The short synthetic peptide sequence of the FLAG-tag (DYKDDDDK) can be removed via cleavage by enterokinase.

Question 55

Glutathione S-transferase (GST)

Answer 55

a 26kDa protein which can is used in GST-pull down assays to purify GST-fused recombinant proteins.

Question 56

6xHis-tag

Answer 56

an oligohistidine domain that will bind to a matrix containing nickel ions (Ni2+) chelated to nitriloacetic acid-agarose.

Question 57

Affinity tags serve 2 other functions besides allowing highly specific purification

Answer 57

Solubilization tags

Used for visualization via staining or western blot

Question 58

Solubilization tags

Answer 58

Are used for recombinant proteins expressed in chaperone-deficient species (e.g. E. coli) to prevent protein aggregation.

Question 59

Used for visualization via staining or western blot

Answer 59

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

Question 60

Affinity chromatography

Answer 60

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

Question 61

6xHis or poly-histidine tag system

Answer 61

Flow-through run on SDS-PAGE

Lane 1: starting material
Lane 2: column flow-through
Lane 3: wash 1
Lane 4: elution

Question 62

illustration of 6xHis Tag system

Answer 62

matrix - spacer arm - nitrilotriacetic acid and nickel form oligohistidine domain - binds to multiple proteins

Question 63

6xhis tag system

Answer 63

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]

Question 64

Affinity chromatography

Maltose-binding protein (MBP) tag

Answer 64

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.

Question 65

Steps involved in amylose affinity chromatography

Answer 65

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.