Cell Purification

Question 1

Objective(s) of the following steps in protein purification:

1. Collection of cells by centrifugation and resuspended in 500 ml buffer A (50 mM Tris-HCl
   pH 7.9) plus 4 mg/ml of lysozyme
2. Incubate for 15 mins at room temperature

Answer 1

1. To break cells

2. Ensure complete breakage of cells by lysozyme

Question 2

Objective(s) of the following steps in protein purification:

3. Transfer the lysis mixture into plastic bottles, and centrifuge at 8000 rpm for 15 minutes at
   4C
4. Apply the supernatant to a DEAE column

Answer 2

3. Remove cell debris to obtain supernatant

4. Separate proteins according to their charges (DEAE is a positively charged resin)

Question 3

Objective(s) of the following steps in protein purification:

5. Wash column with 6 volumes of buffer B (10mM Tris-HCL pH 7.9)
6. Elute column in a single step with buffer C (10mM Tris-HCL pH 7.9, 200mM KCI)

Answer 3

5. Wash out positively charged proteins (pI > 7.9)

6. Elute out the negatively charged proteins (pI < 7.9)

Question 4

How would you purify a recombinant protein by employing affinity chromatography?

Answer 4

1. Resuspend cell pellet in wash buffer (Buffer with low conc. imidazole)
2. Use ultrasonication to disrupt the cell
3. Spin down cell lysate in centrifuge to separate supernatant from cell debris.

(Optional: The supernatant is applied to an ultrafiltration unit – to reduce the volume of supernatant so that the protein mixture is concentrated. (if ultrafiltration is included)

4. Equilibrate the column with binding buffer (Sodium Phosphate Buffer alone)
5. Load supernatant into Ni-NTA column, carry out centrifugation to ensure His-Tag protein binds to resin
6. Load column with wash buffer (Buffer with low imidazole conc), carry out centrifugation, to wash out non-specific proteins
7. Load column with elution buffer (Buffer with high imidazole conc), carry out centrifugation, to elute His-tag proteins
8. Apply molecular exclusion column to the eluted fraction to remove imidazole

Question 5

What is sodium phosphate NaH2PO4 used for?

Answer 5

It is a basic buffer solution used in chromatography

Question 6

What is imidazole?

Answer 6

Imidazole has high affinity with the Ni-NTA resin.

It can be used in high conc for elution buffer, as it will out-compete the His-tag protein for binding to the resin.

It can also be used at a lower conc for wash buffers, to elute non-target proteins that weakly bind (unspecific binding) to the resin.

Question 7

What is potassium chloride, KCI used for?

Answer 7

It will increase the ionic strength of the buffer.

Question 8

What is the role of glycerol in the SDS-PAGE sample loading buffer?

Answer 8

Increase density of protein samples

Question 9

What is the role of reducing agent in the SDS-PAGE sample loading buffer?

Answer 9

To break the disulfide bonds, allowing the protein to unfold, so that protein will be sorted through the gel by molecular weight

Question 10

What is the role of SDS in the SDS-PAGE sample loading buffer?

Answer 10

Bind onto protein molecules, rendering protein molecules all negatively charged.

Disrupt intra-molecular non-covalent bonds in protein molecules, allowing protein to unfold further

Question 11

What is the role of tracking dye in the SDS-PAGE sample loading buffer?

Answer 11

To visualise the movement of the protein as it moves down the gel

Question 12

What are examples of a reducing agent?

Answer 12

Mercaptoethanol, DTT

Question 13

Describe how pH control can be achieved in bioreactors used for mammalian cell
cultivation.

Answer 13

Sodium bicarbonate added to the culture media provides bicarbonate ions.
NaHCO3 Na+ + HCO3-

By increasing the partial pressure of CO2 in the bioreactor headspace, additional H+ ions can be produced, thus lowering the pH value.

CO2 CO2 + H2O H2CO3 H+ + HCO3-

By decreasing the partial pressure of CO2 in the bioreactor headspace, bicarbonate ions will shift the equilibrium reaction to the left, increasing the pH value.

Question 14

Explain the difference in pH regulation between bacterial fermentation
and mammalian cell cultivation.

Answer 14

Strong acid (such as HCl) and strong base (such as NaOH) are used for pH regulation in bacterial fermentation

Sodium bicarbonate and CO2 are used for pH regulation in mammalian cell cultivation.

Question 15

State the difference between a perfusion bioreactor and a continuous
bioreactor

Answer 15

Perfusion bioreactor has cell retention device. Continuous bioreactor does not have such a device.

Question 16

Explain why a perfusion bioreactor is advantageous to mammalian cell expression system but not advantageous to bacterial cell expression system

Answer 16

Mammalian cells produce extracellular protein product. Thus the protein product
is in the supernatant and harvested in a perfusion bioreactor. The returned
mammalian cells can continue to produce extracellular protein product, resulting
in higher productivity.

However, bacterial cells produce intracellular protein product, thus no protein
product is harvested from the outlet stream in a perfusion bioreactor.

Question 17

Explain why a continuous bioreactor with feedback system is employed for bacterial expression system

Answer 17

A continuous bioreactor with feedback systems is to limit the exit of biomass or
to return a portion of the concentrated biomass to the bioreactor.

This is advantageous because biomass concentration can be increased and
substrate concentration can be decreased in bioreactor. This is to fully utilise the
substrate.

Question 18

What are some disadvantages of serum use?

Answer 18

Serum is chemically undefined, uncharacterized and has varied composition. It cannot ensure consistent cell growth performance.

Serum may create potential contamination with viruses and other infectious agents.

Serum is expensive

Presence of proteins complicate downstream processing of desired biopharmaceutical

Question 19

Explain how you would examine the protein expression level during a bacterial
fermentation

Answer 19

O: At each specific growth rate, ensure fermentation is at steady-state before taking cell sample

1. Take a cell sample from the bioreactor
2. Centrifuge the cell sample
3. Resuspend the cell pellet for cell disruption
4. Centrifuge the cell extract to obtain the supernatant
5. Mix the supernatant with sample loading buffer, and heat it up
6. Load protein sample into SDS-PAGE gel for gel electrophoresis
7. After gel electrophoresis, stain gel and detain with proper solutions
8. Scan gel to obtain the protein expression level

Question 20

When should cells be induced with IPTG? Why?

Answer 20

Log phase. This is to ensure that cells are viable and able to express foreign gene. It is also to achieve high cell density.

Question 21

Why is it important to use SDS in the sample loading buffer?

Answer 21

To ensure the protein is denatured and covered with negative charges

Question 22

Suggest four factors that may affect protein expression level in E. Coli glycerol stock culture with antibiotic that is induced with IPTG.

Answer 22

1. Deletion of foreign gene
2. Low IPTG concentration
3. Low copy number of foreign gene
4. Cells not induced in the log phase

Question 23

Explain transient expression of protein

Answer 23

When plasmid DNA did not integrate with chromosome DNA, resulting in decreasing specific productivity over time

Question 24

Column regeneration is the same as column reequilibration. True or false?

Answer 24

True

Question 25

Explain how ion exchange chromatography can be conducted to purify a recombinant protein further

Answer 25

9. Load the first fraction to DEAE column to separate proteins according to their charges
10. Load wash buffer (sodium phosphate buffer) to DEAE column (which is positively charged) to wash out positively charged proteins with pI >8
11. Load elution buffer (buffer with KCL) to elute negatively charged proteins with pI < 8, including the recombinant protein
12. Apply molecular exclusion column to the eluted fraction to remove KCL

Question 26

List four sensors which are commonly used in bioreactors for monitoring
fermentation parameters.

Answer 26

Temperature sensor

pH sensor

Dissolved oxygen sensor

Foam sensor

Level sensor

Question 27

Why is pH control for bacterial cell cultivation systems not suitable for mammalian cells?

Answer 27

Mammalian cells may not be able to tolerate strong acid and base used for pH regulation in bacterial fermentation.

Question 28

Explain why mammalian cell cultivation requires bioreactors with a much larger
volume compared with those required for bacterial fermentation

Answer 28

Difficult to achieve high cell density for mammalian cell culture
compared with bacterial cell culture

Difficult to achieve the same expression level of protein product
in mammalian cells as that in bacterial cells.

Thus bioreactors with much
larger volume are required in order to obtain large amount of protein
molecules

Question 29

List three factors that limit the scale-up of the cell cultivation process

Answer 29

Mass transfer limitation from gas to liquid

Shear damage to cells from mixing

Toxic metabolites build-up that inhibit cell growth

Question 30

Serum media is recommended for mammalian cell cultivation on a large scale. True or false?

Answer 30

False. Serum-free media should be used for large-scale mammalian cell cultivation.