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CHE 541 > 14 - Protein Purification > Flashcards

14 - Protein Purification Flashcards

1
Q

What is protein purification?

A

Better fraction of protein (enrichment)

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2
Q

Why would someone purify proteins?

A
  1. Study them (activity, structure)

2. Use them (drugs, etc.)

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3
Q

How come human antibodies are hard to make in E. coli?

A

They need post translational modifications (glycosylation)

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4
Q

What is a major issue with protein purification?

A

The source, and how much you start with

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5
Q

How is molecular biology useful for protein purification?

A

Can overexpress a protein to make it more abundant

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6
Q

Using overexpression, how much of a protein product can be produced (relative to everything else)?

A

40-60%

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7
Q

What are issues with protein purification (getting to the lysate)?

A

Expression, lysis, solubilization, stabilization, etc.

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8
Q

What are some examples of expression systems?

A

E. coli, yeast, insects, and mammalian cells

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9
Q

What type of cells often use overexpression?

A

E. coli (not mammalian cells)

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10
Q

What are some strategies for protein purification?

A

Solubility, charge, polarity, size, and ligand-binding affinity

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11
Q

True or false: you can enrich a protein using either physical or chemical properties

A

True: these can both be utilized

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12
Q

What is solubility a function of?

A

Salt, pH, and temperature

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13
Q

For solubility, what is usually altered?

A

Salt content

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14
Q

What is the basis of solubility?

A

Multiple acid/base groups, which need favorable ionic bonds

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15
Q

What is an important parameter for solubility?

A

Ionic strength

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16
Q

What is the general shape of solubility in salt?

A

Initial rise, then a fall in solubility

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17
Q

True or false: all salts change solubility the same

A

False: each salt changes solubility differently

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18
Q

What is salting in?

A

Salt is added to increase solubility

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19
Q

How does salting in increase solubility?

A

More counterions shield residues, reducing protein/protein interactions

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20
Q

What is salting out?

A

Salt is added to decrease solubility

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21
Q

How does salting out decrease solubility?

A

Competition between salt ions and water molecules, leading to protein aggregation

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22
Q

How do (NH4)2SO4 cuts work?

A

Gradually increase ammonium sulfate concentration, and different proteins have different solubilities (can pick particular protein)

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23
Q

What is the advantage of ammonium sulfate cuts?

A

Cheap and easy to do (large quantities of proteins)

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24
Q

What is the disadvantage of ammonium sulfate cuts?

A

Not very high purity

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25
Q

What are some examples of polarity purification?

A

Chromatography and non-polar stationary phase

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26
Q

What is fractionation?

A

A mobile phase (liquid) with a stationary phase (solid)

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27
Q

What is reverse phase chromatography?

A

Use a hydrophobic stationary phase

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28
Q

Which species elute first in reverse phase chromatography?

A

Polar species (don’t interact with stationary phase)

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29
Q

Which species elute last in reverse phase chromatography?

A

Nonpolar species (interact with stationary phase)

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30
Q

What is the disadvantage with reverse phase chromatography?

A

It denatures the protein (not useful if you need folded protein)

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31
Q

What does HPLC stand for?

A

High performance liquid chromatography

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32
Q

What is the advantage of HPLC?

A

High resolution (sharp peaks) and fast separation

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33
Q

When is silica gel used, and why?

A

Normal phase, because it has a lot of hydroxyls to interact with polar molecules

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34
Q

How come reverse phase is used over normal phase?

A

Solubility and mobile phase

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35
Q

What does the mobile phase work better with reverse phase?

A

Water is polar, so need to differentiate

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36
Q

What determines how a protein interacts with the solid phase?

A

Free energy differences (how quickly is it bound and unbound)

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37
Q

What detector is usually used for HPLC?

A

UV or mass

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38
Q

How is UV used for HPLC?

A

Measure Trp or Tyr

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39
Q

What is the problem with UV in HPLC?

A

Need to absorb to be seen (usually ok)

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40
Q

What is the problem with mass spec in HPLC?

A

Often do not have full proteins, need to make assumptions

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41
Q

True or false: HPLC can use only one of UV or mass

A

False: it can use both

42
Q

How can proteins be separated by charge?

A

Using ion exchange chromatography

43
Q

How does ion exchange chromatography work?

A

A charge solid phase is used, and protein binding will occur based on charge

44
Q

What materials is HPLC made out of and why?

A

Metals, because of high pressures

45
Q

What materials is ion exchange made out of and why?

A

Glass, because of lower pressures

46
Q

What is the advantage of ion exchange chromatography over HPLC?

A

Lower pressures, so the protein will not get denatured

47
Q

How can charge be changed in the mobile phase?

A

pH, buffer salt, salt concentration, etc.

48
Q

What happens if salt is increased in the mobile phase?

A

Breaks interactions between protein and solid phase (change free energy)

49
Q

True or false: cellulose exchange is only for positive exchange

A

False: it can be used for both positive or negative

50
Q

What are the requirements for a stationary phase in ion exchange chromatography?

A

Charged, does not denature protein, look similar to water, insoluble in mobile phase, does not change shape

51
Q

How are proteins eluted in ion exchange chromatography?

A

Add salt to change free energy, and cause proteins to elute

52
Q

What materials are good for the stationary phase in ion exchange chromatography?

A

Sugars and polymers (cellulose, dextrans, agarose, polystyrene)

53
Q

What solvent is used for ion exchange chromatography?

A

Water

54
Q

What solvent is used for HPLC?

A

Mixed solvents

55
Q

For anion exchange, what is the charge on the stationary phase?

A

Positive

56
Q

For cation exchange, what is the charge on the stationary phase?

A

Negative

57
Q

How can cellulose be used in both cation and anion exchange?

A

It can have differently charged functional groups

58
Q

What cellulose group is used for cation exchange?

A

CM (carboxymethyl, negatively charged)

59
Q

What cellulose group is used for anion exchange?

A

DEAE (diethylaminoethyl, positively charged)

60
Q

What is pI?

A

pH where protein has equal positive and negative charges

61
Q

If pI < pH 7, which ion exchange should be done and why?

A

Anion exchange (protein is negative)

62
Q

If a protein is positively charged, which ion exchange should be done?

A

Cation exchange (attract positive protein)

63
Q

If a protein is negatively charged, which ion exchange should be done?

A

Anion exchange (attract negative proteins)

64
Q

If pI > pH 7, which ion exchange should be done and why?

A

Cation exchange (protein is positive)

65
Q

If cation exchange is used, what amino acids are present on the protein?

A

Lys (positive protein)

66
Q

If anion exchange is used, what amino acids are present on the protein?

A

Asp and Glu (negative protein)

67
Q

What does the stationary phase look like for size exclusion chromatography?

A

Has matrix with pores of different sizes

68
Q

Which species elute first in size exclusion chromatography and why?

A

Larger species, because they don’t enter the beads (less distance)

69
Q

Which species elute last in size exclusion chromatography and why?

A

Smaller species, because they go through the pores (more distance)

70
Q

What is the resolution of size exclusion chromatography based on?

A

The size of the pores

71
Q

Besides enrichment, what are some other uses for size exclusion chromatography?

A

Can estimate molecular weight, and desalt

72
Q

What is the composition of the stationary phase in size exclusion chromatography?

A

Polysaccharides

73
Q

How come size exclusion chromatography cannot exceed 5-10% of column volume?

A

Avoid saturation of beads, smaller proteins will also go through

74
Q

How much material can be collected from size exclusion chromatography?

A

mg of material

75
Q

How can better resolution or protein enrichment be achieved?

A

By using a two stage strategy (two different chromatographies)

76
Q

What does dialysis separate species by?

A

Large size differences

77
Q

What two species could be separated by dialysis?

A

Protein and salt

78
Q

How does dialysis work?

A

Diffusion across a membrane of certain speices

79
Q

What is the problem with dialysis?

A

Low resolution (need big size differences)

80
Q

For small quantities, what can be used for size separation?

A

Gel electrophoresis

81
Q

What is a 2D gel?

A

SDS-PAGE to separate by size and pI

82
Q

What is a 2D gel useful for?

A

Protein identity (soak out peptides and run mass spec to confirm)

83
Q

What is the difference between affinity chromatographies and other chromatographies?

A

Affinity chromatographies is a chemical property, while the others (size exclusion, ion, etc.) are physical properties

84
Q

What does a graph of a biophysical property enrichment look like?

A

Lots of peaks with different properties

85
Q

What does a graph of an affinity property look like?

A

A huge peak (wash), and a small peak (what you are interested in)

86
Q

What type of enrichment strategy is used for mammalian systems more commonly and why?

A

Affinity, because there are low concentrations (vast majority is wash)

87
Q

What are the three types of affinity chromatography?

A

Immobilized metal chromatography, maltose binding protein, and immunoprecipitation

88
Q

How does metal affinity work?

A

His can coordinate with metal, and thus will interact with the solid phase

89
Q

What is needed for a protein to bind with the solid phase in metal affinity chromatography?

A

A 6X-His tag

90
Q

How is a 6X-His tag added to a protein?

A

It is cloned and added to the N or C terminus

91
Q

How can proteins with the His6 tag be eluted from the column?

A

Add imidazole (competition), or drop pH (protonate imidazole so it cannot coordinate)

92
Q

What are the advantages of His6 chromatography?

A

Small His6 tag, cheap resin, high capacity, can be native or denature

93
Q

What are the disadvantages of His6 chromatography?

A

Can interfere with protein-protein interactions (protease cleavage site), higher background than other affinity purifications (other metal binding motifs)

94
Q

What does MBP stand for?

A

Maltose binding protein

95
Q

How does MBP chromatography work?

A

MBP is added to a protein, so it can interact with the resin

96
Q

What are the advantages of MBP chromatography?

A

More specific than His6, milder elution conditions, can increase protein yield,

97
Q

What are the disadvantages of MBP chromatography?

A

Can interfere with many assays, need to be cleaved, need cloning

98
Q

How does antibody chromatography work?

A

Antibodies already have affinity complex for a protein

99
Q

How are antibodies secured in the chromatography?

A

They can be immobilized, or free (and captured later)

100
Q

What is the advantage of antibody chromatography

A

Very specific (10,000x enrichment)

101
Q

What are the disadvantages of antibody chromatography?

A

Need antibodies, expensive, need low pH (harsh)

102
Q

What is the difference between biophysical separation and affinity separation?

A

Affinity separation has better purification, but is more complicated (need genetic constructs)

CHE 541 (20 decks)

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