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AP0609 Component 1 > Protein purification > Flashcards

Protein purification Flashcards

1
Q

What is a recombinant protein?

A
  • Gene of interest is cloned into an expression plasmid
  • Plasmid is transferred to host cell
  • High levels of the protein can be produced in the host cell
  • The protein can be purified for functional studies
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1
Q

What are the advantages of protein expression in E.Coli?

A
  • Fast growth rate
  • Can transform bacteria with plasmid DNA rapidly
  • Relatively cheap
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2
Q

What are the disadvantages of protein expression in E.coli

A
  • Proteins may not fold correctly
  • High concentration of protein can be insoluble
  • Lack some post translational modifictions
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3
Q

What is the process of extracting proteins from bacteria?

A
  • Lyse the cells without denaturing or degrading the protein using freeze-thaw, non-ionic detergent or sonication
  • Centrifuge again after lysis - the supernatant contains soluble cellular material, including proteins
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4
Q

Define protein purification

A

To exploit a specific properrty of the protein e.g. size, charge, affinity tag, hydrophobicity, biological activity, in order to purify and enrich your target protein

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5
Q

What do you need to consider when purifying a protein?

A
  • recovery
  • Cost
  • Purity
  • Functionality
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6
Q

What are the different methods of protein purification?

A
  • Differential solubility
  • Affinity chromatography
  • Size exclusion chromatography
  • Ion exchange chromatography
  • Hydrophobic interaction chromatography
  • Isoelectric focusing
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7
Q

Throughout protein purification how do you track the progression?

A

SDS-PAGE and Western blotting/immunoblotting

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8
Q

What is the principle of SDS-PAGE?

A
  • Protein samples are loaded into wells of gel and electric current is applied
  • negatively charged proteins travel towards the positive electrode
  • Proteins move down the gel according to size - the smaller proteins travel further
  • Proteins are visualized using Coomassie blue
  • Checks the purity of the sample
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9
Q

What is the principle of Western blotting?

A

Allows the specific detection of protein of interest after SDS-PAGE
- Proteins are transferred to a membrane and incubated with specific antibodies which can be tagged

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10
Q

What is the principle of differential solubility?

A
  • Often used as an initial purification step
  • Techniques including salting out, polyethene glycol, heat and altering pH
  • Precipitated protein can then be re-dissolved and subject to additional purification
  • Polar water molecules interact with the hydrophilic regions of protein - increasing the solubility
  • Anything that affects protein charge, protein structure or protein water interactions will affect protein solubility
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11
Q

How does the protein folding affect its solubility?

A
  • Proteins are folded such that charges/polar amino acids are on the surface of the protein (hydrophilic) and hydrophobic amino acids are hidden inside the structure
  • Proteins are solubilized by hydrogen bonding with polar water
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12
Q

How does ammonium sulphate cause protein precipitation?

A
  • Addition of high salt concentration leads to displacement of the water molecules and precipitation of the protein
  • Water bonds with the salt rather than the protein
  • Proteins bind to each other and precipitate
  • Different proteins have different solubility in aqueous solution
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13
Q

Why do we use ammonium sulphate in salt precipitation?

A
  • Highly water soluble
  • Cheap
  • Available at high purity
  • No permanent denaturation of the proteins
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14
Q

Why do you need to consider next step after salt precipitation?

A
  • the salt may need to be removed before the next purification step - for example cant perform ion exchange straight after
  • however you can perform gel filtration and hydrophobic interaction chromatography straight after
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15
Q

What are the three ways you can remove the salt after salt precipitation

A
  • Dialysis
  • Gel filtration chromatography
  • Diafiltration
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16
Q

What is the principle of dialysis?

A
  • Sample is placed in a bag with a semi permeable membrane
  • Can choose permeability based on target protein
  • After a few changes of the buffer the salt will be completely removed
17
Q

What is the priciple of gel filtration in terms of salt purification?

A
  • Seperates components based on size
  • Resins have pores that some small molecules can enter
  • Load dissolved protein and salt onto collumn and flush sample through with buffer
  • Small salt ions enter the pores of the resin, whilst larger molecules i.e. proteins travel straight through, therefore salt moves through a lot slower and can be seperated
18
Q

What is the principle of diafiltration?

A
  • Pressure driven filtration membrane
  • slat passes through the membrane whilst the protein is retained in the sample
19
Q

What dictates a proteins charge?

A
  • The number of amino acid side chains in the protein that can donate or gain H+
20
Q

Are charged amino acids hydrophobic or hydrophillic?

A

Hydrophilic

21
Q

What is the isoelectric point

A

The point at which the protein has no overall net charge

22
Q

How does heat denaturing cause protein precipitation?

A
  • Unfolding of proteins exposes the inner hydrophobic amino acids that tend to bind to each other , causing proteins to bind to each other and precipitate
  • Some proteins are thermostable (above 45 degrees) therefore you can use heat to precipitate out the contaminating proteins
23
Q

What is the principle of affinity chromatography?

A
  • Uses and affinity resin composed of an affinity molecule and a solid support
  • The affinity matrix specifically recognizes the protein of interest.
  • Cell lysate is incubated with the affinity resin, protein of interest binds the the resin and you can wash away the other proteins. The purified protein can then be eluted from the beads.
24
Q

What is an example of affinity chromatography?

A

The purification of a protein with a His Tag using a affinity matrix of Nickel

25
Q

What are the 2 types of tags used in affinity chromatography?

A
  • Epitope tag - for protein identification
  • Affinity tag - for protein purification
26
Q

What is gel filtration chroatography also known as?

A
  • Size exclusion chromatography
27
Q

What is the principle of size exclusion chromatography?

A
  • Separates based on size
    Column packed with porous resin
  • Can be completed with a high salt content
  • Large proteins exit the column quicker than small proteins
  • The elution volume is the volume of buffer at which the protein of interest exits the column
  • You take a sample from each fraction to perform a western blot
28
Q

What are the factors that effect gel filtration chromatography?

A
  • Size/mass of protein
  • Shape of protein
  • Length of column - longer gives better separation
  • Amount of protein
  • Resin material
  • Protein complexes
29
Q

Describe the principle of ion exchange chromatography

A
  • Separates the proteins based on charge
  • at a pH below the pKa, sid chain accepts H+
  • At a pH above pKa, side chain loses H+
  • Overall protein charge can be changed by increasing or decreasing pH
  • pH below the isoelectric point - protein is net positive
  • pH above the isoelectric point - protein is net negative
  • If you know the pI you can adjust the pH to alter net protein charge
  • The resin has either a positive or negative charge
  • cation (negative charge) binds to positive proteins
  • Anion (positive charge) binds to negative proteins
  • Bound proteins are then eluted using increasing salt solution which compete for the ionic interaction therefore displace the proteins.
30
Q

Describe the principle of Hydrophobic interaction chromatography

A
  • interaction between hydrophobic patches of a protein and resin coated in hydrophobic material
  • This happens in aqueous solution
  • Loaded in a column with high salt buffer
  • this displaces the water and reveals the hydrophobic patches
  • Salt concentration is inversely proportional to protein hydrophobicity
  • decreasing salt concentration used for elution of the protein
31
Q

What are the factors that effect protein elution in HIC?

A
  • Choice of salt in buffer
  • Include non-ionic detergents
  • Reduce temp
  • change in pH
32
Q

Describe the principle of isoelectric focusing

A
  • Isoelectric point is when the protein has no overall net charge
  • protein is loaded onto a gel with a pH gradient
  • An electric field is applied and protein migrates based on their charge
  • They stop migrating when they reach PI
33
Q

What can effect protein migration in isoelectric focusing?

A

Phosphorylation

34
Q

What are the two factors that are exploited during 2 dimensional electrophoresis

A

Size
pH

35
Q

What might cause poor protein concentration?

A
  • Poor protein expression in bacteria
  • Inefficient lysis
  • Inefficient purification
  • Inefficient elution
  • Protein is insoluble
  • Protein degradation
36
Q

How can you minimize protein degradation?

A
  • main cause is protease enzymes being released during lysis
  • work quick
  • work over ice
  • include protease inhibitors in solution
  • Monitor with SDS-PAGE
37
Q

How would you calculate specific enzyme activity (U/mg)

A

enzyme activity (U) / total protein (mg)

38
Q

How would you calculate yield (%)

A

enzyme activity after purification step / enzyme activity in original sample * 100

39
Q

How would you calculate enrichment factor?

A

specific activity after purification step / specific activity in original sample

AP0609 Component 1 (7 decks)

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