Lecture 20 - Using Recombinant DNA Technologies to Make Proteins

Question 1

The genetic code is____?

Answer 1

Universal

Question 2

_____ reverse transcribes into ______ to generate a DNA sequence that doesn’t contain introns

Answer 2

mRNA, cDNA

Question 3

What are the key steps in producing a recombinant protein? (5)

Answer 3

Isolate, Clone, Transform, Grow, Purify

1. Isolate gene of interest
2. Clone into expression vector
3. Transform into bacteria for expression or isolation of more DNA for use in another expression system.
4. Grow cells expressing protein of interest in appropriate expression system
5. Isolate and purify protein

Question 4

What is Preproinsulin processed into?

Answer 4

Proinsulin via signal peptide cleavage

Question 5

What is Proinsulin process into?

Answer 5

Mature insulin via C-Peptide cleavage

Question 6

What 2 chains are left in the mature insulin produced?

Answer 6

A and B chains

Question 7

How are the A and B chains held together?

Answer 7

Via disulphide bonds

Question 8

Where is insulin produced and where is it fuether processed?

Answer 8

produced in the pancreas, further produced in the golgi. (does not happen in bacteria)

Question 9

What is the first step in recombinant insulin production?

Answer 9

Obtaining human insulin cDNA (via reverse transcription where intron are removed)

Question 10

What is the second step in recombinant insulin production?

Answer 10

Close gene into expression vectors (two vectors are needed, one for A and B subunits)

Question 11

What are needed within the recombinant vectors?

Answer 11

• Bacterial promoter
• Origin of replication
• Antibiotic resistance gene

Question 12

What is the third step in recombinant insulin production?

Answer 12

Transform bacteria (into seperate colonies)

Question 13

What is the fourth step in recombinant insulin production?

Answer 13

Grow bacteria expressing insulin A and B chains

Question 14

What is the fifth step in recombinant insulin production?

Answer 14

Extraction and purification of viral vectors from sample.

Question 15

What is the sixth and last step in recombinant insulin production?

Answer 15

Combining of A subunit and B subunit vectors via disulphide bonds.

Question 16

What method was first used to create disulphide bonds?

Answer 16

Air oxidiation

Question 17

Advantages of prokaryotic systems:

Answer 17

• relatively low cost
• high yield
• pathogen free

Question 18

Disadvantages of prokaryotic systems:

Answer 18

• Proteins often partially folded
• inability to preform stable post-translational modifications.

Question 19

What does recombinant insulin using mammalian cells allow?

Answer 19

• Protein can be produced as pre-pro-protein and processed efficiently
• The protein would be secreted from cells (easier purification)
• More expensive.

Question 20

What would be the steps in making recombinant insulin in eukaryotic cells instead of bacterial?

Answer 20

1. Isolate cDNA for insulin.
2. clone into eukaryotic expression vector
3. Transform bacteria to produce more vector DNA
4. Transfect eukaryotic cells
5. Extract recombinant insulin and purify

Question 21

Whats the most appropriate system for Glycosylation and Folding?

Answer 21

Mammalian

Question 22

What does EPO stand for?

Answer 22

Erythropoietin

Question 23

What is the structure of EPO?

Answer 23

• 4 helix bundle (simple and easy to fold)
• Post translationally modified (glycosylation)
• Made in mammalian cells due to PTM

Question 24

rEPO is made in what animal cells?

Answer 24

Chinese Hamster Ovary cells. (CHO)

Question 25

Where is EPO produced?

Answer 25

The kidneys

Question 26

What does EPO act to do?

Answer 26

Stimulate bone marrow to produce red blood cells. (lack of EPO can lead to anemia)

Question 27

What does glycosylation of EPO do to the structure?

Answer 27

- increases the stability in bloodstream - Required for proper biological function - Helps the protein solubilize in the blood stream - Ensures appropriate immune recognition.

Question 28

What types of cells are recombinant human EPO produced in?

Answer 28

Mammalian cells (due to need of PTM)

Question 29

What is the process of expression of the rhEPO vector?

Answer 29

1. Eukaryotic expression vector needed. 2. Specific promoter for transcription in mammalian cells 3. Purification and administration. 4. Injected into bloodstream always.

Question 30

How is EPO considered a performing enhancing drug?

Answer 30

Due to increase in RBCs, this leads to an increase in the oxygenation of muscle. More ATP is generated from oxygen in muscle burning sugar and fats.

Question 31

Is rhEPO and EPO the same?

Answer 31

No, rhEPO is designed to mimic natural EPO, but has cells that add sugars in a manner that can differ significantly from humans.

Question 32

What method can be used to test for rhEPO and EPO?

Answer 32

Isoelectric focusing. (IEF)

Question 33

How does Isoelectric focusing work?

Answer 33

Separates proteins based on their isoelectric point. Due to rhEPO and EPO have different isoelectric points, a visual difference will appear due to gel moving patterns.

Question 34

What is pharming?

Answer 34

Using a whole animal to make recombinant proteins.

Question 35

Do all cells in culture perform all PTMs equally?

Answer 35

No, an example being Gamma Carboxylation (PTM)

Question 36

What was the first protein produced for a transgenic animal?

Answer 36

Antithrombin (AT)

Question 37

What does Antithrombin do?

Answer 37

Acts as a natural blood thinner to prevent blood clotting.

Question 38

Where is the AT protein expressed?

Answer 38

In the milk of transgenic goats at lactation

Question 39

What is the process in reproducing the AT protein?

Answer 39

Promotor and AT are inserted into egg, then into goat, offspring produced.