3. Affinity Chromatography

Question 1

What is the general principle?

Answer 1

Ligand immobilized on matrix. Biospecific interactions (which can be ionic, hydrophobic, or both) between ligand and target proteins. Unwanted proteins flow through.

Question 2

What are the different types of general ligand-target combos?

Answer 2

Ab-antigen; ligand-receptor; substrate-enzyme.

Question 3

List the steps

Answer 3

1. Equilibration
2. Load sample
3. Wanted proteins bind, unwanted flow through.
4. Wash with buffer to rid of excess unbound proteins.
5. Elute bound proteins by changing buffer conditions (pH, ionic strength).
6. Reequilibration.

Question 4

Why do we equilibrate and reequilibrate?

Answer 4

Equilibration: ensures buffer condition is equivalent to that of protein conditions to prevent protein denaturation or precipitation.
Reequilibration: allows for the reuse of the column by washing with buffers containing denaturants and chaotropes to remove residual bound proteins, preventing contamination.

Question 5

What are the characteristics of the matrix and give examples.

Answer 5

1. Physically withstand high pressure.
2. Chemically withstand harsh conditions like pH changes.
   Eg. Sepharose: cross-linked agarose that resists denaturation.

Question 6

What are the characteristics of the ligand and give examples.

Answer 6

1. Highly specific to target protein and easily coupled to matrix.
2. Able to form stable and reversible complexes with target proteins.
3. Able to dissociate easily from proteins without any adverse biochemical changes.
   Eg. Small molecules like glutathione; macromolecule like protein A (group specific), lectin, Abs (monospecific)

Question 7

What is a common issue in AC?

Answer 7

Steric hindrance

Question 8

Explain the common issue in AC.

Answer 8

It is when proteins are not able to bind optimally to the ligand due to their size or conformation.

Question 9

Explain the solution to the common issue in AC.

Answer 9

Use spacer arms which extend the ligand away from the matrix, allowing proteins to bind optimally to the ligand.

Question 10

What is another issue with the solution to the common problem in AC and how do we fix it?

Answer 10

Spacer arms consist of inert hydrophobic chain structures that can form non-specific hydrophobic interactions. So we have to reduce the length of the spacer arms as much as possible.

Question 11

How do we couple the ligand to the matrix? Give an example and explain.

Answer 11

Via derivatisation. Like cyanogen bromide activation. Hydroxyl groups are activated by CNBr and add to surface of matrix where they form cyanate esters, allowing for amino-group containing ligands to covalent bind with the matrix.

Question 12

What are the 4 ways to elute proteins?

Answer 12

1. Change buffer pH
2. Change buffer ionic strength
3. Use denaturants
4. Use competitors (eg. Free reduced glutathione displaces GST-bound fusion proteins)

Question 13

List the 3 elution methods, their conditions, and principles.

Answer 13

1. Step elution, stepwise change: conditions of buffer change at the end of the process where column is free of contaminants and only bound proteins are left.
2. Gradient elution, linear change: conditions of buffer change gradually throughout the process where weakly bound proteins elute first.
3. Isocratic elution, no change in conditions.

Question 14

Compare the peaks of gradient and isocratic elutions.

Answer 14

Gradient results in narrower peaks which indicate better separation and resolution. Also results in a more complete elution hence more frequent peaks.
Isocratic has wider peaks. Only target proteins get detected.

Question 15

What are the 3 ligand-target combos?

Answer 15

1. Streptavidin-biotin
2. Protein A/G-Ig
3. Lectin-glycan

Question 16

Explain streptavidin-biotin.

Answer 16

Proteins get biotinylated and then loaded. Biotin region of proteins interact with streptavidin which is the ligand.

Question 17

Explain protein A/G-Ig.

Answer 17

Proteins become fusion proteins with Ig binding domains which interacts with protein A/G which is the ligand.

Question 18

Where do protein A and G come from?

Answer 18

A comes from S. aureus. G comes from Streptococcal bacteria.

Question 19

What human and mouse subclasses do proteins A/G bind to?

Answer 19

All human Ig subclasses and all mouse IgG subclasses except for IgA, IgM, and serum albumin.

Question 20

What are lectins?

Answer 20

Plant-derived macromolecules that bind to sugars.

Question 21

What are lectin-glycans used for?

Answer 21

Purifying glycoproteins and differentiating terminal sugars in glycans.

Question 22

State the different types of lectins, their source, what they bind to, and the eluting sugar.

Answer 22

1. Concanavalin A; jackbean seeds; a-D-mannose and a-D-glucose; a-D-methylmannose.
2. Wheat germ agglutinin; wheat germs; N-acetyl-b-D-glucosamine and N-acetyl-b-D-neuraminic acid; N-acetyl-b-D-glucosamine.
3. Pisum sativum lectin; peas; a-D-mannose; a-D-methylmannose.
4. Soybean lectin; soybean; N-acetyl-b-D-galactosamine; N-acetyl-b-D-galactosamine.

Question 23

How can fusion proteins be purified in affinity chromatography?

Answer 23

Epitope tags can be attached to proteins upstream or downstream to form fusion proteins where the ligand interacts with the tag and the tag can also be enzymatically cleaved to recover native protein.

Question 24

What are the 5 types of tags?

Answer 24

1. Hemagglutinin (HA) tag
2. FLAG tag
3. Glutathione-S-transferase (GST) tag
4. 6xHis tag
5. Maltose-binding protein (MBP) tag

Question 25

Explain each tag.

Answer 25

1. HA tag: a short sequence of polypeptide from influenza virus.
2. FLAG tag: a short synthetic sequence of peptide that can also be cleaved by enterokinase.
3. GST tag: a 26kDa protein that purifies GST-bound recombinant proteins via GST pull-down assays.
4. 6xHis tag: contains histidine residue which interacts with nitrilotriacetic acid as the ligand with nickel ions on matrix.
5. MBP tag: MBP tag attached to proteins which interacts with amylose as the ligand. Amylose/maltose can also be used to elute and cleave protein.

Question 26

What are the other 2 functions of tags and give examples.

Answer 26

1. Solubilize proteins of chaperone deficient species like E. coli. Eg. Thioredoxin can be used as solubilization tag.
2. Visualization during staining/WB/ELISA. Eg. Enzyme-linked anti-HA and anti-FLAG Abs.

Question 27

List the steps of amylose affinity chromatography.

Answer 27

1. Clone and express POI as fusion protein with an engineered proteolytic cleavage site.
2. Load it into amylose affinity column.
3. MBP tag region of protein binds to amylose on the matrix.
4. Elute fusion protein with amylose/maltose.
5. Cleave fusion protein with amylose/maltose to obtain native protein.