14 - Protein Purification Flashcards
What is protein purification?
Better fraction of protein (enrichment)
Why would someone purify proteins?
- Study them (activity, structure)
2. Use them (drugs, etc.)
How come human antibodies are hard to make in E. coli?
They need post translational modifications (glycosylation)
What is a major issue with protein purification?
The source, and how much you start with
How is molecular biology useful for protein purification?
Can overexpress a protein to make it more abundant
Using overexpression, how much of a protein product can be produced (relative to everything else)?
40-60%
What are issues with protein purification (getting to the lysate)?
Expression, lysis, solubilization, stabilization, etc.
What are some examples of expression systems?
E. coli, yeast, insects, and mammalian cells
What type of cells often use overexpression?
E. coli (not mammalian cells)
What are some strategies for protein purification?
Solubility, charge, polarity, size, and ligand-binding affinity
True or false: you can enrich a protein using either physical or chemical properties
True: these can both be utilized
What is solubility a function of?
Salt, pH, and temperature
For solubility, what is usually altered?
Salt content
What is the basis of solubility?
Multiple acid/base groups, which need favorable ionic bonds
What is an important parameter for solubility?
Ionic strength
What is the general shape of solubility in salt?
Initial rise, then a fall in solubility
True or false: all salts change solubility the same
False: each salt changes solubility differently
What is salting in?
Salt is added to increase solubility
How does salting in increase solubility?
More counterions shield residues, reducing protein/protein interactions
What is salting out?
Salt is added to decrease solubility
How does salting out decrease solubility?
Competition between salt ions and water molecules, leading to protein aggregation
How do (NH4)2SO4 cuts work?
Gradually increase ammonium sulfate concentration, and different proteins have different solubilities (can pick particular protein)
What is the advantage of ammonium sulfate cuts?
Cheap and easy to do (large quantities of proteins)
What is the disadvantage of ammonium sulfate cuts?
Not very high purity
What are some examples of polarity purification?
Chromatography and non-polar stationary phase
What is fractionation?
A mobile phase (liquid) with a stationary phase (solid)
What is reverse phase chromatography?
Use a hydrophobic stationary phase
Which species elute first in reverse phase chromatography?
Polar species (don’t interact with stationary phase)
Which species elute last in reverse phase chromatography?
Nonpolar species (interact with stationary phase)
What is the disadvantage with reverse phase chromatography?
It denatures the protein (not useful if you need folded protein)
What does HPLC stand for?
High performance liquid chromatography
What is the advantage of HPLC?
High resolution (sharp peaks) and fast separation
When is silica gel used, and why?
Normal phase, because it has a lot of hydroxyls to interact with polar molecules
How come reverse phase is used over normal phase?
Solubility and mobile phase
What does the mobile phase work better with reverse phase?
Water is polar, so need to differentiate
What determines how a protein interacts with the solid phase?
Free energy differences (how quickly is it bound and unbound)
What detector is usually used for HPLC?
UV or mass
How is UV used for HPLC?
Measure Trp or Tyr
What is the problem with UV in HPLC?
Need to absorb to be seen (usually ok)
What is the problem with mass spec in HPLC?
Often do not have full proteins, need to make assumptions
True or false: HPLC can use only one of UV or mass
False: it can use both
How can proteins be separated by charge?
Using ion exchange chromatography
How does ion exchange chromatography work?
A charge solid phase is used, and protein binding will occur based on charge
What materials is HPLC made out of and why?
Metals, because of high pressures
What materials is ion exchange made out of and why?
Glass, because of lower pressures
What is the advantage of ion exchange chromatography over HPLC?
Lower pressures, so the protein will not get denatured
How can charge be changed in the mobile phase?
pH, buffer salt, salt concentration, etc.
What happens if salt is increased in the mobile phase?
Breaks interactions between protein and solid phase (change free energy)
True or false: cellulose exchange is only for positive exchange
False: it can be used for both positive or negative
What are the requirements for a stationary phase in ion exchange chromatography?
Charged, does not denature protein, look similar to water, insoluble in mobile phase, does not change shape
How are proteins eluted in ion exchange chromatography?
Add salt to change free energy, and cause proteins to elute
What materials are good for the stationary phase in ion exchange chromatography?
Sugars and polymers (cellulose, dextrans, agarose, polystyrene)
What solvent is used for ion exchange chromatography?
Water
What solvent is used for HPLC?
Mixed solvents
For anion exchange, what is the charge on the stationary phase?
Positive
For cation exchange, what is the charge on the stationary phase?
Negative
How can cellulose be used in both cation and anion exchange?
It can have differently charged functional groups
What cellulose group is used for cation exchange?
CM (carboxymethyl, negatively charged)
What cellulose group is used for anion exchange?
DEAE (diethylaminoethyl, positively charged)
What is pI?
pH where protein has equal positive and negative charges
If pI < pH 7, which ion exchange should be done and why?
Anion exchange (protein is negative)
If a protein is positively charged, which ion exchange should be done?
Cation exchange (attract positive protein)
If a protein is negatively charged, which ion exchange should be done?
Anion exchange (attract negative proteins)
If pI > pH 7, which ion exchange should be done and why?
Cation exchange (protein is positive)
If cation exchange is used, what amino acids are present on the protein?
Lys (positive protein)
If anion exchange is used, what amino acids are present on the protein?
Asp and Glu (negative protein)
What does the stationary phase look like for size exclusion chromatography?
Has matrix with pores of different sizes
Which species elute first in size exclusion chromatography and why?
Larger species, because they don’t enter the beads (less distance)
Which species elute last in size exclusion chromatography and why?
Smaller species, because they go through the pores (more distance)
What is the resolution of size exclusion chromatography based on?
The size of the pores
Besides enrichment, what are some other uses for size exclusion chromatography?
Can estimate molecular weight, and desalt
What is the composition of the stationary phase in size exclusion chromatography?
Polysaccharides
How come size exclusion chromatography cannot exceed 5-10% of column volume?
Avoid saturation of beads, smaller proteins will also go through
How much material can be collected from size exclusion chromatography?
mg of material
How can better resolution or protein enrichment be achieved?
By using a two stage strategy (two different chromatographies)
What does dialysis separate species by?
Large size differences
What two species could be separated by dialysis?
Protein and salt
How does dialysis work?
Diffusion across a membrane of certain speices
What is the problem with dialysis?
Low resolution (need big size differences)
For small quantities, what can be used for size separation?
Gel electrophoresis
What is a 2D gel?
SDS-PAGE to separate by size and pI
What is a 2D gel useful for?
Protein identity (soak out peptides and run mass spec to confirm)
What is the difference between affinity chromatographies and other chromatographies?
Affinity chromatographies is a chemical property, while the others (size exclusion, ion, etc.) are physical properties
What does a graph of a biophysical property enrichment look like?
Lots of peaks with different properties
What does a graph of an affinity property look like?
A huge peak (wash), and a small peak (what you are interested in)
What type of enrichment strategy is used for mammalian systems more commonly and why?
Affinity, because there are low concentrations (vast majority is wash)
What are the three types of affinity chromatography?
Immobilized metal chromatography, maltose binding protein, and immunoprecipitation
How does metal affinity work?
His can coordinate with metal, and thus will interact with the solid phase
What is needed for a protein to bind with the solid phase in metal affinity chromatography?
A 6X-His tag
How is a 6X-His tag added to a protein?
It is cloned and added to the N or C terminus
How can proteins with the His6 tag be eluted from the column?
Add imidazole (competition), or drop pH (protonate imidazole so it cannot coordinate)
What are the advantages of His6 chromatography?
Small His6 tag, cheap resin, high capacity, can be native or denature
What are the disadvantages of His6 chromatography?
Can interfere with protein-protein interactions (protease cleavage site), higher background than other affinity purifications (other metal binding motifs)
What does MBP stand for?
Maltose binding protein
How does MBP chromatography work?
MBP is added to a protein, so it can interact with the resin
What are the advantages of MBP chromatography?
More specific than His6, milder elution conditions, can increase protein yield,
What are the disadvantages of MBP chromatography?
Can interfere with many assays, need to be cleaved, need cloning
How does antibody chromatography work?
Antibodies already have affinity complex for a protein
How are antibodies secured in the chromatography?
They can be immobilized, or free (and captured later)
What is the advantage of antibody chromatography
Very specific (10,000x enrichment)
What are the disadvantages of antibody chromatography?
Need antibodies, expensive, need low pH (harsh)
What is the difference between biophysical separation and affinity separation?
Affinity separation has better purification, but is more complicated (need genetic constructs)
