Protein Purification

Question 1

What is protein purification?

Answer 1

Multi-step fractionation process to isolate a single protein from a crude mixture

Question 2

What is the most challenging job as a biochemist?

Answer 2

Protein purification

Question 3

What was the first enzymes purified and crystalized?

Answer 3

Urease and Pepsin

Question 4

What are the steps for protein purification?

Answer 4

1. Cell lysis
2. Differential centrifugation
3. Salt fractionation
4. Dialysis
5. Ion-exchange
6. Gel filtration
7. Affinity
8. Concentration
9. Purified protein which will go through structural and functional analysis

Question 5

What is cell lysis?

Answer 5

Disruption of cells

Physical methods - mechanical shearing

Chemical methods - solubilizing plasma membranes

Enzymatic methods - cell wall digestion

Question 6

What is cell fractionation?

Answer 6

Using centrifugation (applying centrifugal field), will isolate high density proteins into a pellet and low density protein as the supernatant

Size and density of particle, duration of centrifugation

Bacteria has less organelles so fractionation is simple but eukaryotic have many organelles.

Question 7

What is differential centrifugation (subcellular fractionation)?

Answer 7

RPM between small and large will stay constant which is bad but RCF will change.

RCF important factors:

• angular velocity (speed)
• average radius

Question 8

What is zonal centrifugation?

Answer 8

Fractionate into bands

• the large particles rapidly sediment
• the more spherical particles rapidly sediment
• influenced by the density of particles and the medium

vacuoles never reach bottom because equal density

Question 9

What is salt fractionation?

Answer 9

1. Protein dissolved in water
   - proteins are soluble because it is polar and makes hydrogen bonds
2. Salt (precipitate and concentrate protein)
3. Protein in ammonium sulfate
   - water divided from protein to ions
   - need to aggregate with each other to bind

Question 10

Less soluble proteins require how much % of salt saturation?

Answer 10

Less soluble proteins require less % of salt saturation.

Question 11

What is the ammonium sulfate precipitation equation?

Answer 11

Weight of salt (g) = Gsat (S2-S1) / 1-(PS2)

Question 12

What is dialysis?

Answer 12

A separation technique of removal of small molecules from a protein mixture using semipermeable membrane

Question 13

What are the uses of dialysis?

Answer 13

Desalting
- removal of salt and many other small molecules from a protein mixture

Removal of small peptides and proteins

• anything under molecular cut-off (MWCO) such as 10 kDA will leave the bag
• anything above will stay inside the bag

Change to fresh buffer to remove more salt due to osmosis

Question 14

Is changing the buffer multiple times better for dialysis?

Answer 14

Yes

Question 15

What are the positively charged amino acids?

Answer 15

Lysine (Lys, K)

Arginine (Arg, R)

Histidine (His, H)

Question 16

What are the negatively charged amino acids?

Answer 16

Aspartic acid (Asp, D)

Glutamic acid (Glu, E)

Question 17

When a protein is coated with positive charges, which ion exchange to use?

Answer 17

Use a carboxymethyl (CM) sepharose which is a cation exchanger with negatively charged beads

Question 18

When a protein is coated with negative charges, which ion exchange to use?

Answer 18

Use a diethyl amino ethyl (DEAE) sepharose which is a anion-exchanger with positively charged beads

Question 19

What are the three properties that affect ion-exchange chromatography?

Answer 19

Charge state of peptide of protein

pH of buffer

pI value, need to be 2.5 pH higher or lower than pI

Question 20

What is cation exchange chromatography?

Answer 20

Negative charged beads in the column

Negative charged proteins are collected in the flow through

Positive charged proteins will bind to the beads

Elution buffer is used to elute the bounded proteins

Question 21

How does separation of proteins depend upon net charge?

Answer 21

With an anion-exchange chromatography (DEAE, + beads), the most positive charge will elute first as it won’t bind and the most negative will tightly bind and elute last

Question 22

What is required to elute proteins?

Answer 22

Salt

Protein binds at low ionic strength and elutes differentially by increasing salt concentration

Question 23

What is gel filtration?

Answer 23

Separation based on size (molecular weight)

Largest molcules are excluded from the beads and elute first

Smallest molecules are included in the beads and elute last

Question 24

What is elution volume (Ve)?

Answer 24

the volume of a buffer required to elute a compound from the time of its injection to its exit from a column

Measures how well a compound interacts with a stationary phase

Question 25

What is the retention factor (K’)?

Answer 25

K’ = Elution volume of a compound (Ve) / Elution volume for a solute which does not interact with stationary phase (void Vo)

Question 26

What can the retention factor (K’) determine?

Answer 26

Determine the molecular weights of unknown proteins

Question 27

What is resolution?

Answer 27

Ability of a column to separate two components (proteins) wide apart

Peaks elute at different volume and do not overlap

Resolution equal or higher than 2 is considered significant

Higher resolution means better separation

Question 28

What does resolution depend on?

Answer 28

Particle size

Flow rate

Column length and diameter

Sample volume