Lecture 9: Protein Purification 2

Question 0

Describe size exclusion chromatography.

Answer 0

Also known as gel filtration or molecular sieve chromatography.
Separation is based on differences in protein mass and shape.
Protein mixture added to a column containing fine, porous beads made of either dextran polymers, agarose or polyacrylamide.
As solution passes through the column, smaller molecules move into the beads and thus are caught/slowed down. Larger molecules cannot enter the beads and so pass through the column faster.
The solution is fractioned. Early fractions contain the largest molecules, while the smallest molecules appear in the latest fractions.

Question 1

Describe chromatography as a technique for protein purification.

Answer 1

Chromatography separates proteins based on:

• size: size exclusion chromatography
• charge: ion exchange chromatography
• hydrophobicity: hydrophobic chromatography
• binding affinity: affinity chromatography

Question 2

Describe ion exchange chromatography.

Answer 2

Column containing beads with a positive or a negative charge.
Proteins are separated according to arrangement of charges on their surface.
Proteins move through the column at rates determined by their net charge at the pH in the column. If the column contains cat ionic exchangers, proteins with a more negative charge move through faster and elute earlier.
Bound proteins are eluted by gradual addition of excess salt solution.

Question 3

Describe hydrophobic interaction chromatography.

Answer 3

Based of the surface hydrophobicity of protein.
Column matrix contains hydrophobic groups which interact with the hydrophobic regions on the protein.
Bound to column in high ionic strength buffer. Hydration of salt ions attracts water molecules away from protein, increasing the hydrophobicity of proteins.
Proteins are eluted by decreasing ionic strength and addition of detergent or solvent.

Question 4

Describe affinity chromatography.

Answer 4

Protein mixture is added to column containing a polymer bound ligand specific for the protein of interest.
Unwanted proteins are washed through the column (fraction early)
Protein of interest is eluted by ligand solution. Ligand may be an antibody, a protein or drug.

Question 5

Describe high performance (pressure) liquid chromatography.

Answer 5

Uses small bead size for superior resolution.
Uses increased flow rate for improved fractionation speed.
Sample
Can be automated.
Higher cost: low-volume, high value proteins. Used as preservative step in industrial scale purification of insulin and IL-2.

Question 6

List methods of protein quantification.

Answer 6

UV-absorption
Calorimetic assays
Enzyme immunoassay

Question 7

Describe UV absorption for the quantification of proteins.

Answer 7

Side chain Absorbance is 280nm, peptide bond Absorbance is 205nm.
Shines UV light of known wavelength through a protein solution, and a detector measures the amount of light that passes through. The difference is the Absorbance of the protein solution.
Can then compare the Absorbance of the sample to a standard curve of the absorbances of known protein concentrations.
Sensitivity is ~10ug/mL.

Question 8

Describe the use of colourimetric assays for the quantification of proteins.

Answer 8

LOWRY METHOD
Complexes of copper protein are reduced by Folin’s reagent. Absorbance is measured at 750nm. Sensitivity ~10ug/mL.
BRADFORD METHOD
Uses principle of Absorbance change consequent on protein dye binding. Absorbance is measured at 595nm, and has sensitivity of ~0.5ug/mL.
A standard curve is used to determine sample concentration.

Question 9

Describe the use of enzyme immunoassay as a means of protein quantification.

Answer 9

Uses the specificity of antibodies.
Highly sensitive
ELISA results measured using a specialised spectrophotometer called an ELISA reader.
1. The surface of the wells of an ELISA dish are coated with the sample.
2. Unoccupied sites on the surface are blocked with a nonspecific protein.
3. Dish is incubated with primary antibody against specific antigen of interest.
4. Dish is then incubated with antibody-enzyme complex that binds the primary antibody.
5. Substrate is added, which leads to the formation of a coloured product, indicating the presence of specific antigen.
Concentration of antigen/protein is determined by ELISA reader. More colour = more protein of interest.

Question 10

Describe how protein purification can be monitored.

Answer 10

SDS-PAGE can be used to quantitatively evaluate protein purification. Used to monitor purification procedure at each stage. Protein is pure whe only one band is detected of the gel.
Other methods can evaluate purification via protein function. Enzymes can be quantified by specific activity. Functional assays can be used hormones, transport proteins and binding proteins.

Question 11

Describe electrophoresis.

Answer 11

Separates proteins by charge and size. Separation by charge is opposed by viscosity and pore size of the medium used.
Shape changes (eg post-translational modifications) may alter mobility.
Mainly used as an analytical technique. Gives approximate molecular weight, number of proteins present or degree of purity.

Question 12

Describe the visualisation of proteins after electrophoresis.

Answer 12

After electrophoresis, proteins must be stained in order to be visualised.
Proteins are fixed in the gel, the SDS is washed out, dye added and then the gel destained. Usually Coomasie Blue or Amido Black stains are used. Silver staining may be used as a highly sensitive alternative.
The stained gel is then compared with a standard containing proteins of known molecular weights.

Question 13

Describe isoelectric focusing.

Answer 13

Separates proteins based on their acidic and basic residues.
An ampholyte solution is incorporated into a gel, and a stable pH gradient is achieved after application of an electric field. Protein solution is added, and an electric field is reapplied. Proteins migrate through the gel until they reach a pH that is equal to their own isoelectric point. At this point the net charge on the protein is zero and it stops migrating.
After staining, proteins are shown to be distributed along pH gradient according to their pI values.

Question 14

Describe 2D electrophoresis.

Answer 14

Combines isoelectric focusing with SDS-PAGE.
First dimension (horizontal): separation of proteins by IEF
Second dimension (vertical): separation of proteins by SDS-PAGE
obtains very high resolution separation of proteins.

Question 15

Describe immunoblotting.

Answer 15

Used to detect a specific protein in a mixture of proteins.

1. Proteins are transferred from SDS gel to a porous membrane via blotting.
2. Blot is probed with a primary antibody specific to protein of interest
3. Blot is then probed with an enzyme-conjugated secondary antibody specific for the primary antibody.
4. Enzyme substrate is added, and the protein can then be visualised via chromogenic detection. This is a semi-quantitative technique.

Question 16

What is proteolysis degradation and how can it be avoided?

Answer 16

Proteolysis degradation is the degradation of proteins by proteases. Proteases belong to 1 of 6 mechanistic classes:
Serine proteases I (mammals) or II (bacteria)
Cysteine proteases
Aspartic proteases
Metalloproteases I (mammals) and II (bacteria).
Proteolysis degradation can be avoided by minimising processing time, processing at lower temperatures and by using protease inhibitors (eg EDTA inhibits metalloproteases)

Question 17

What can be added to a purified therapeutic product to stabilise proteins? How do they work?

Answer 17

Some Amino acids (eg glycine), carbohydrates (eg glucose) and polyols (eg glycerol) have been found to be good protein stabilisers. They work by stabilising protein conformation (eg against heat), reducing surface adsorption of product, or by inhibiting aggregate formation.