Protein Purification

Question 1

What are the features of protein surface that can be exploited during purification?

Answer 1

Residual hydrophobicity, extensive charged patches and ligand binding sites.

Question 2

Why are proteins purified?

Answer 2

Allows study of enzyme function. Allows study of protein-protein/protein-nucleic acid interactions. Necessary for structural analysis (high amounts of pure protein especially needed for XC)

Question 3

What is salting out useful for?

Answer 3

Fractionating a mixture of proteins and concentrating dilute solutions of proteins.

Question 4

What is the Hofmeister series?

Answer 4

Ranks ions on their ability to precipitate out a mixture of hen egg white proteins.

Question 5

What happens when an organic solvent is added to a protein solution?

Answer 5

An ordered layer of water forms at the surface of a hydrophobic region on the surface of the protein.

Question 6

Describe the process of salting out.

Answer 6

Dissolve protein mix in high conc. ammonium sulfate. Divalent ions excluded from ordered water layer, lowering the entropy of the system. Addition of divalent ions at a specific concentration will disrupt the ordered water. Protein sequesters its hydrophobic core and is precipitated.

Question 7

What are the two types of protein chromatography?

Answer 7

Adsorption/desorption and permeation

Question 8

What are is the difference between adsorption/desorption and permeation chromatography?

Answer 8

Adsorption/desorption is solid-liquid phase and is dependent on the thermodynamics of the interaction between protein and the solid phase. Permeation is liquid-liquid phased and is dependent on the rate of diffusion between liquid phases. There is no interaction between the liquids and the solid phase.

Question 9

What needs to be considered when designing a chromatography experiment?

Answer 9

Selectivity of the stationary phase, non-specific interactions, liquid flow, column design and binding capacity.

Question 10

Why do non-specific interactions need to be considered when designing chromatography experiments?

Answer 10

Occur due to residual hydrophobicity and charge interactions- decrease the resolution of the method.

Question 11

Why does the adsorption of the target protein to the solid phase need to be highly reversible under controlled conditions?

Answer 11

Need to be able to control when the target protein binds and dissociates from the solid phase.

Question 12

Give reasons as to why the protein mixture cannot be flowed through the column too quickly.

Answer 12

Protein needs a certain amount of time in contact with the solid phase in order to reach equilibrium. Fast flow causes turbulent flow which affects dispersion.

Question 13

Why can the protein mix not be flowed through the column too slowly?

Answer 13

Increases diffusion- need a balance between establishing equilibrium and preventing dispersion.

Question 14

What are the causes of protein band dispersion?

Answer 14

Diffusion and Turbulent flow.

Question 15

What are the main things to consider when choosing a host cell for expression?

Answer 15

• target protein prokaryotic or eukaryotic?
• target protein post translationally modified?
• sensitivity to host cell proteases?
• localisation of the target protein

Question 16

What is the Langmuir Isotherm used to describe?

Answer 16

The equilibrium of a soluble protein binding to a solid phase.

Question 17

What does good adsorption depend on?

Answer 17

High concentration of binding sites and a low dissociation constant.

Question 18

Give the range of alpha values for adsorption and desorption.

Answer 18

0.8-1 for adsorption and less than 0.5 for desorption

Question 19

How can α be manipulated?

Answer 19

By manipulating Kd - by changing buffer, pH, ionic strength.

Question 20

Why is α a crude measure of binding?

Answer 20

Adsorption sites are not of equal strength, there is actually always a range of Kd values. Total protein concentration is not constant within a protein band. Manufactured surfaces assume there is 1 binding events, if the protein had more than one, its affinity for the column would increase. Concentration of binding sites varies.

Question 21

Describe diffusion in adsorption/desorption chromatography.

Answer 21

Regions at the back and ahead of the protein band contain no protein- molecules therefore diffuse into these regions, creating a diffusion gradient. Band spreads as it moves through the column. Diffusion increases with column length and is inversely proportional to the Mw of the protein.

Question 22

As a result of band spreading, what shaped profile do proteins elute as?

Answer 22

Gaussian profile (bell shaped)

Question 23

Describe turbulent flow in adsorption/desorption chromatography.

Answer 23

Solution flows through channels between particles of the solid phase. Particles and channels are uneven, meaning that turbulent backflow occurs and lowers resolution of the method.

Question 24

How can an adsorption/desorption method be refined?

Answer 24

By calculating the optimal flow rate.

Question 25

What does optimal rate increase with?

Answer 25

Increasing column length.

Question 26

How are narrower protein bands achieved?

Answer 26

By using finer particles in the solid phase- less turbulent flow. By flowing at optimal rate and minimising diffusion.

Question 27

What is the self-sharpening effect?

Answer 27

Diffusion occurs as soon as flow starts, affecting protein concentration in different regions of the protein band. At low protein conc. the protein is more likely to adsorb meaning migration down the column slows in comparison to high protein conc. Results in sharpening of the leading edge and an extended elution profile for the trailing edge.

Question 28

What is a homogeneous sample?

Answer 28

A pure sample containing only one protein species.

Question 29

How can you tell if a purification method is effective?

Answer 29

Purify a protein using multiple purification methods, and measure the specific activity after each purification. The method that causes the biggest increase in specific activity is the most effective.

Question 30

What needs to be taken into consideration when choosing a purification method?

Answer 30

Whether you need to prioritise purity (e.g. for XC) or yield depending on what you want to do with the protein next. Efficiency of the purification method.