Lecture 20

Question 1

What are the key steps in producing recombinant proteins?

Answer 1

Isolate gene of interest, clone into expression vector, transform into bacteria for expression or isolate of more DNA for use in another expression system, grow cells expression protein of interest in appropriate system, isolate and purify protein

Question 2

Why is insulin expression different?

Answer 2

Because it contains two chains (A and B) which are connected by disulfide bonds and it is also processed in the golgi

Question 3

What is the solution to expression insulin in bacterial cells?

Answer 3

Express the A and B chain separately

Question 4

When do the A and B chains get combined to form insulin?

Answer 4

In the purifying step

Question 5

What are the advantages of using a prokaryotic system

Answer 5

Relatively low cost, receive a high yield and pathogen-free sample

Question 6

What are the disadvantages of using a prokaryotic system

Answer 6

Proteins often partially fold if folding is complicated, bacterial cells are incapable of performing stable post-translational modifications

Question 7

What are possible other systems to use when prokaryote systems are not sufficient?

Answer 7

Mammalian or transgenic animal systems

Question 8

What makes mammalian systems more efficient?

Answer 8

Proteins can be produced as a pre-pro-protein and processed efficiently, the protein would be secreted from cells making purification easier

Question 9

What is the disadvantage of mammalian systems?

Answer 9

They are very expensive and much slower

Question 10

What would be different in making recombinant insulin in eukaryotic cells

Answer 10

Isolate cDNA for insulin, clone into eukaryotic expression vector, transform bacteria to produce more vector DNA, transfect eukaryotic cells, extract recombinant insulin from cell media, purify insulin

Question 11

What is erythropoietin (EPO)?

Answer 11

A hormone that your kidneys naturally make to stimulate the production of red blood cells

Question 12

Why must rEPO be expressed in a mammalian system?

Answer 12

Because it is post-translationally modified through glycosylation

Question 13

What is glycosylation?

Answer 13

The addition of carbohydrates

Question 14

What mammalian system is rEPO expressed in?

Answer 14

Chinese hamster ovary cells (CHO)

Question 15

Why would we want to make rEPO?

Answer 15

Many disease states result in lowered red blood cell counts, leading to anaemia

Question 16

What diseases can cause anaemia?

Answer 16

Chronic renal failure and cancer treatments (chemo)

Question 17

What does EPO do?

Answer 17

Signals red bone marrow to make more red blood cells

Question 18

Why is the glycosylation of EPO necessary?

Answer 18

Increases stability in the bloodstream, helps protein solubilise, ensures appropriate immune recognition and is required for proper biological functioning

Question 19

How does rEPO enhance exercise performance?

Answer 19

Increases RBC leading to increased oxygenation of the muscle and the generation of more ATP

Question 20

What is the composition of natural EPO?

Answer 20

It has a glycosylation pattern that is very specific to human cells, 4x sugar groups

Question 21

How does rEPO differ from natural EPO?

Answer 21

The CHO cells add sugars in a manner that can differ significantly from human cells

Question 22

How is rEPO detected?

Answer 22

Using isoelectric focusing, separating proteins based on their isoelectric point on a gel with a pH gradient. Proteins stop moving once they reached their pl point.

Question 23

How do the isoelectric points of hEPO and rhEPO differ?

Answer 23

They have different charges due to the different glycosylation patterns, stopping at different pl points

Question 24

What is pharming?

Answer 24

A biotechnology process that involves genetically modifying plants or animals to produce pharmaceutical substances

Question 25

When would pharming be used?

Answer 25

When the post-translational modifications are too complicated for a mammalian cell

Question 26

What is the example protein from pharming?

Answer 26

gamma-carboxylation of glutamate

Question 27

What is gamma-carboxylation of glutamate involved in?

Answer 27

Blood clotting

Question 28

What is the blood clotting drug?

Answer 28

Antithrombin (AT)

Question 29

How is recombinant antithrombin expressed?

Answer 29

In transgenic animals; goats

Question 29

Where is the AT protein expressed in transgenic goats?

Answer 29

In their milk produced during lactation

Question 30

How does the goat become transgenic?

Answer 30

The mammary gland-specific sequence is combined with the AT gene, introduced to somatic cells and fused with a goat egg. The transgenic goat is born and all its offspring will produce the same gene.