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PHAR 505 > Protein Purification > Flashcards

Protein Purification Flashcards

1
Q

There are many reasons to purify protein. What are the 3 reasons discussed in class?

A

Structure determination
Affinity assays
Functional characterization

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2
Q

What natural sources are rich in proteins? What happens if they are not?

A

Sperm whale muscles
Pancreas
Bacteria

If protein is not abundant, large amounts of starting material is required

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3
Q

How can proteins be overexpressed?

A

Via genetically modified systems i.e. E coli, baculovirus, etc

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4
Q

Describe the process of protein expression in E Coli

A
  1. Make plasmid containing the GOI
  2. Transform Plasmid in E. Coli
  3. Grow cells in shaker
  4. Adjust temperature and add inducer for expression of protein
  5. Harvest cells
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5
Q

What are 4 properties of E. Coli plasmids?

A

DNA pol Origin of Replication
Antibiotic resistance (for selection)
Regulated transcription promotor, usually controlled by an inducible operator like the lac operon
A multiple cloning site

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6
Q

Which E coli strains are engineered for protein expression? Why are they engineered?

A

BL21
JM109
C41

They lack proteases that would normally degrade overexpressed protein, and may contain DE3 gene encoding a special polymerase

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7
Q

How are plasmids incorporated into E Coli?

A

Heat shock

Electroporation

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8
Q

What are the 3 pros and 2 cons of Protein expression via E coli?

A

Pros:
Easy and fast
Cheap
Can use minimal medium (for incorporation of NMR isotopes)
Cons:
Heterologous expression (lack mammalian chaperones etc)
Fast translation rate may lead to aggregates “inclusion bodies”

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9
Q

What is Pichia Pastoris?

A

A yeast strain that can use methanol as a sole carbon source

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10
Q

How can P. pastoris be converted to secretion mode?

A

By attaching the alpha mating factor in medium

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11
Q

When would you use P. pastoris over eg E Coli?

A

For secreted glycosylated protein expression

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12
Q

How can you use S. frugiperda to express protein?

A

Isolate clonal cells Sf9 or Sf21 from moths, which are susceptible to infection by baculoviruses (large lytic viruses)

Large “bacmids” need to be generated by homologous recombination with smaller E. Coli plasmids

Co-transfected in insect cells with plasmids containing viral genes

(expensive and lengthy)

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13
Q

What mammalian cells do you use to express protein?

A

Human embryonic kidney cells (HEK293)

Or Chinese Hamster Ovary cells

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14
Q

When would you use Mammalian cells to express protein?

A

Useful for protein overexpression in tissue culture or suspension culture
Idela environment for mammalian protein expression (contains all necessary factors for proper folding)

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15
Q

How do you get transfected cells with plasmids to express transiently?

A

A strong promoter such as Cytomegalovirus

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16
Q

What is an affinity tag and how could it be useful?

A

A region in the gene coding for a protein domain that can easily be filtered out during a purification assay (eg His6, GST, biotin)

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17
Q

Why wouldn’t you put an affinity tag at the start or end of a gene?

A

Could affect function of the protein

18
Q

If you’re adding an affinity tag, what else should you add to make sure the protein behaves properly?

A

Protease site, to remove the affinity tag after purification (Rhinovirus 3C, TEV, Thrombin)

19
Q

What is codon optimization and when is it important?

A

Different codons code for the same amino acid, but some codons are favored in certain organisms.
In the case of heterologous transfections, you may need to optimize your gene to reflect the relevant concentration of tRNA

20
Q

How are cells collected after protein expression is completed?

A

Low-speed centrifugation

21
Q

What are the main components of resuspension medium and what is their function?

A

Buffer (maintain pH) - HEPES or Tris
Salts to maintain solubility (salting in effect)
Detergents (to extract membrane-bound proteins) - CHAPS triton or deoxycholate
Urea to solubilize protein aggregates
Protease inhibitors, EDTA

22
Q

What are 5 ways to lyse cells?

A

Sonication (good for bacteria and mammalian cells)
French cell press (bacteria and yeast)
Freezing-grinding (yeast)
Lysozyme treatment (gram-negative bacteria)
Dounce Homogenizer (mammalian cells)

23
Q

How is debris removed from cells?

A

High speed centrifugation

24
Q

What are the two components to Chromatography, and the key principle that makes it useful?

A 
Mobile phase (buffer) in continuous flow
Stationary phase (resin) with desired chemical properties

The different interactions between the proteins and the solid phase allows for the separation (elution) in the mobile phase

25
Q

What are the 4 main steps in Liquid Chromatography?

A

Equilibrating the resin with buffer
Loading the Sample
Washing off contaminants
Eluting the protein of interest

26
Q

What 4 properties are exploited for protein purification?

A

Affinity
Size/hydrodynamic volume
Electrostatic charge
Hydrophobic interactions

27
Q

How do you purify small-organic molecules or small peptides via liquid chromatography?

A

Reverse-phase chromatography

28
Q

How does affinity chromatography work?

A

It relies on a specific interaction between the target protein and the resin
Elution is carried using high concentrations of a competitor, or low pH to dissociate antibody-ligand interactions
(selective separation of contaminant)

29
Q

Does affinity chromatography require FPLC?

A

No, can be carried out by gravity

30
Q

What is the Beer-Lambert law?

A

A = epsilon x B x C

Where A is the absorbance (2-log%T)
epsilon is the absorption coefficient
B is the path length of the UV cell
C is the concentration

31
Q

How does size-exclusion chromatography work?

A

Beads have pores of different sizes, and proteins elute at different times depending on their size (largest first, smaller later)

32
Q

Why do smaller proteins take longer to elute during SEC?

A

Because they get caught in all the pores of the beads, while larger molecules just have to go around

33
Q

What is the relation between size of a protein and elution time during SEC?

A

Linear

34
Q

How come you can determine quaternary structure during SEC?

A

Because it’s done under native conditions

35
Q

How does SDS PAGE drive proteins?

A

By passing a current that drives the protein:SDS complexes at a rate that is dependent on the charge:mass ratio (larger molecules migrate slower than smaller ones)

36
Q

How does Ion Exhange chromatography work?

A

The stationary phase consists of beads charged with chemical groups that reversibly interact with surface charges of the protein

37
Q

What are the two types of Ion exchange chromatography?

A

Cation-exchange: The resin is negatively charged and binds positively charged proteins
Anion exchange where the resin is positively charged and binds negatively charged proteins

38
Q

How can the net charge of a protein be modulated with pH? (useful for ion exchange chromatography)

A

If pH < pI, the protein will be positively charged

If pH > pI, the protein will be negatively charged

39
Q

What is Hydrophobic interaction chromatography and how does it work?

A

Resin consists of beads with hydrophobic groups that reversibly interact with hydrophobic patches on the protein (enhanced by salts)
Useful for purifying proteins without any affinity tags
Difficult to optimize binding conditions

40
Q

What is reverse-phase chromatography and how does it work?

A

Similar to hydrophobic affinity chromatography
Stationary phase consists of silica beads coupled to non-polar groups
Mobile phase consists of strong organic acid (denatures proteins and peptides, allows for interactions with the non-polar resin)

41
Q

How do you concentrate your protein of interest?

A

Ultrafiltration through a membrane that only lets micromolecules through (centrifuged)

PHAR 505 (11 decks)

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