Protein Purification

Question 1

You cloned a gene of interest with a C-terminal His-tag and expressed the protein in bacteria. How would you purify the protein?

Answer 1

Affinity chromatography would be used in this case, specifically Nickel (Ni2+) immobilised on to magnetic beads as Nickel (Ni2+) binds strongly to His-Tag.

Question 2

What is Western blotting, when would you use it and why?

Answer 2

Western blot is often used in research to separate and identify proteins
A mixture of proteins is separated based on molecular weight, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein.

Question 3

What is Immunoblotting and why would it be performed?

Answer 3

Immunoblotting can be done after a Western blot and involves binding a primary antibody to your protein, then a secondary antibody to that with a fluorochrome attached that can then be detected for fluorescence.

Question 4

What is the principle of Gel filtration?

Answer 4

Protein is passed through agarose gel; larger proteins pass through faster as they do not enter the pores within the gel whereas large proteins can readily avoid these pores and pass through.

Question 5

Gel filtration of a known protein is run and you compare your mass to the standards however your protein does not fall within its known mass what could be the reason for this?

Answer 5

Some proteins exist as Dimer’s and Trimer’s so the kda may be higher than expected.

Question 6

What does SDS PAGE measure and what would a 2D measurement look at?

Answer 6

SDS PAGE is reliable method for determining the molecular weight (MW) of an unknown protein, this can be done on a 2D scale by separating proteins according to their isoelectric points.

Question 7

What are the advantages of using bacteria for protein production?

Answer 7

• Fast growth rate (20min doubling time) – can generate lots of protein-expressing bacteria very quickly
• Can transform bacteria with plasmid DNA rapidly (less than 5 minutes)
• Relatively cheap

Question 8

What are the advantages of using bacteria for protein production?

Answer 8

• Proteins may not fold correctly
• High concentration of protein can be insoluble (inclusion bodies)
• Lack some post-translational modifications (e.g. phosphorylation)

Question 9

What are methods of Lysing bacterial cells expressing target protein? What should you do after the cells have been lysed?

Answer 9

• Freeze-thawing (e.g. in liquid N2)
• Non-ionic detergent (e.g. Triton X-100)
• Sonication (ultra-high frequency sound)

Step 2- Centrifuge again after cell lysis – supernatant contains soluble cellular material, including proteins

Question 10

What is the principle behind differential solubility using Ammonium Sulphate Precipitation?

Answer 10

Proteins having terminals with charges (NH3+ and COO-) these interact with H20 as H has a slight positive charge and O a slight negative. Due to these charges’ proteins fold into structures within water with the hydrophilic layer on the outside of the cell membrane, these interactions increase solubility.

Addition of Ammonium Sulphate Disrupts the polar interactions of the protein and the H2O leading to aggregates forming and decreasing solubility. Water bonds with salt ions instead of proteins & proteins bind with hydrophobic areas on other proteins and precipitate.

Question 11

How would you use differential solubility using Ammonium Sulphate Precipitation to select for a specific protein?

Answer 11

Different proteins have different solubilities in aqueous solution, by changing the amount of salt added we can solubilise specific proteins.

Question 12

Why use Ammonium Sulphate for precipitation?

Answer 12

• Highly water-soluble
• Relatively cheap
• Available at high purity
• No permanent denaturation of proteins (e.g., enzymes will remain active)

Question 13

What are 3 most common methods for removing salt from your protein sample?

Answer 13

• Dialysis
• gel filtration chromatography
• diafiltration

Question 14

How does Dialysis remove salt from the protein solution?

Answer 14

1.Sample is placed in a bag with semi-permeable membrane (‘pores’)
2.Choose permeability based on target protein
3. Pores too small to allow passage of your protein but big enough to allow passage of salt ions (salt reaches equilibrium)
4.Several changes of buffer eventually remove the salt from your sample

Question 15

How does gel filtration chromatography remove salt from the protein solution?

Answer 15

 Load dissolved protein (and salt) onto column – flush sample through with buffer
 Resin has pores/holes that some components can enter
 Small salt ions enter the pores of resin, whilst large proteins pass straight through (carried in the buffer)

Question 16

How does Diafiltration remove salt from the protein solution?

Answer 16

1. Sample is passed through a cycle, with buffer constantly flowing.
2. At one end of the cycle a permeable filtration module is present which allows salt to pass through
3. New buffer is constantly added in this cycle until salt is fully removed
    Salt passes through membrane (permeate)
    Protein is retained in sample (retentate)

Question 17

What is the principle of using heat denaturing to purify protein sample?

Answer 17

Heat denatures the tertiary structure of proteins exposing hydrophobic interior causing aggregation (hydrophobic binding to each other). If your protein is thermos stable at a certain temperature, you can heat to this temperature and either remove your protein as precipitate or soluble

Question 18

What is the principle of using affinity chromatography to purify protein sample?

Answer 18

1. Affinity resin/matrix composed of an affinity molecule bound to a solid support – e.g., Sepharose beads
2. Affinity matrix specifically recognises protein of interest
3. Protein may be engineered to have specific tag
4. When mixed with cell extract target protein should bind to affinity resin
5. Beads can then be centrifuged and washed, removing unbound extract components (batch purification)
6. Purified target protein can then be eluted from beads

Question 19

How would you take measurements from Gel filtration?

Answer 19

Collect fractions over time based on size and Measure elution volume - the volume of buffer at which a particular protein exits the column

Question 20

What factors effect Gel separation chromatography?

Answer 20

 Size/mass of protein - in effect, the molecular radius, which is generally proportional to mass
 Shape of protein (e.g. globular vs fibrous)
 Correct Gel type as certain resins are specific for their weight ranges
 Length of column (some columns >1m long!) – longer columns give better separation
 Amount of protein – too much protein can cause broad elution peaks

Question 21

What is the iso-electric (pl) point and how does this effect protein charge?

Answer 21

The pH at which a protein has no net charge.

When the protein is an an environment lower than the Iso-electric point it accepts protons becoming more positive & vice versa

Question 22

What is the principle of Ion Exchange Chromatography (IEC)

Answer 22

IEC separates proteins based on charge
that comes from ionisation of amino acid side chains (form ions via loss or gain of H+)

Sample is passed through a column with ‘ ion exchange resin beads’
Cation exchange resin – Negatively charged cellulose resin bead that binds with positively charged Cations

Bound proteins are then eluted with buffer containing increasing salt concentration

Salt ions compete for ionic interactions and displace proteins

Question 23

What is the principle of Hydrophobic Interaction Chromatography (HIC)

Answer 23

Interaction between hydrophobic patches on protein and resin coated in Hydrophobic material

Proteins have hydrophobic patches which are covered by H20 in aqueous solution

In HIC, sample is prepared and loaded onto column in high salt buffer (e.g. ammonium sulphate)

This displaces water and exposes hydrophobic patches allowing binding of hydrophobic patches on resin and protein

Question 24

How is salt concatenation related to hydrophobicity within Hydrophobic Interaction Chromatography (HIC)

Answer 24

As hydrophobicity increases a larger concentration of salt is required.

Question 25

What is the principle of Isoelectric Focusing (IEF)

Answer 25

Proteins with different pIs can be purified/separated using IEF
Protein is loaded into Gel with a stable gradient of PH, and an electric current is passed through.

The protein passes through the gradient towards the positive and negative electrodes depending on its charge until it reaches its (Pl) where it is stable and can be separated.

Question 26

How would you check the purity of your protein.

Answer 26

Analyse purity using SDS-PAGE.

Question 27

What might cause low protein concentration in final solution of protein purification ?

Answer 27

 Poor protein expression in bacteria – optimise growth/IPTG
 Inefficient lysis – try other methods/combinations
 Inefficient purification – reduce detergent/salt
 Inefficient elution – optimise
 Protein is insoluble – optimise expression conditions/use mammalian host
 Protein degradation - proteins are prone to degradation throughout the process

Question 28

What might minimise the chance of Protein Degradation during purification?

Answer 28

 Low temperature – keep reagents on ice, work in cold room
 Work quickly
 Include protease inhibitors
 Include chelating agents (e.g. EDTA) – bind metal ions that are needed for protease activity preventing degradation of Dna
 Include buffers to avoid acidification

Question 29

What is the calculation for Yield?

Answer 29

Yield (%) = enyme activity after purification step / enzyme activity in original sample
x 100 (for %)

Question 30

What is the calculation for enrichment factor?

Answer 30

specific activity after purification step
/ specific activity in original sample

Question 31

What is the calculation for Specific enzyme activity?

Answer 31

Specific enzyme activity (U/mg) = enzyme activity (U) / total protein (mg)

Question 32

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