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Question 1

Key steps in producing a recombinant protein (brackets are yappage) - JELLY FISH EXAMPLE

Answer 1

1. Isolate gene of interest (isolate mRNA and make cDNA (mature GFP) using reverse transciptiase)
2. Clone into expression plasmid (need restiction sites that are complementary between plasmid vector and the gene we wanna put in, plasmid has R1 sites available, need R1 sites either side of our GFP - using PCR, make primers that are complementary to GFP (amplifying GFP), but have R1 sites off each end, then have complementary sticky ends used to paste GFP into vectors - DNA ligase)
3. Transform into bacteria for expression or isolation of more DNA for use in another expression system (uses chemical transformation or elceltroporatoin) (bacteria amplyifys the plasmid - lots of copies using the Origin or replication and DNA polymerase )
4. Grow cells expressing a protein of interest (antibiotic plate - only bacteria that will grow is the bacteria with plasmid with specific gene)( then put into liquid flask for more growing of bacteria containing plasmid
5. Isolate and purify the protein

Question 2

Key steps in procuring a recominnt protein

Answer 2

1. Isolate gene of interest
2. Clone into expression plasmid - which one?
3. Transform (chemical/electricalporation) into bacteria for expression or isolation of more DNA for use in another expression system
4. Growing cells expressing protein of interest
5. Isolate and purify the protein

Question 3

Recombinant insulin production - producing cDNA
yappage

Answer 3

C- chain holds A and B chain together, after a single polypeptide is produced the C chain is then cleaved out

• DNA is transcribed into mRNA after splicing to remove the introns - mRNA is then reverse transcribed into cDNA that is then cloned into plasmid
  -cDNA contains its swignal, b chain, c chain and a chain

Question 4

Problem with insulin being produced in bacteria and solution

Answer 4

• insulin is produced in the pancreas as a pre-proprotein that is further processed by golgi
• c chain is cleaved by proteases in normal eukaryotic cells but bacteria doesn’t have proteases the there is no way to produce mature insulin
• instead we produce the A and B chains separately in two different bacterial
• instead of full length cDNA we pcr amplify a cDNA copy that just contains the b chain or the a chain
• these can now be transformed to bacteria
• then purify a and b
• then mix back together in a one to one ratio under conditions that allow disulfide bonds to be formed again

Question 5

Signal sequence in insulin is required for

Answer 5

Secreition

Question 6

In a mature protein the a and b chains are held together by…

Answer 6

Disulphides bonds with intervening C -chains - chain must be removed

Question 7

Purpose of c chain

Answer 7

Ensure equal amounts of a and b chain

Question 8

Clone gene into expression plasmids and transform bacteria Arising problem with inserting two individual chains into bacteria and solution

Answer 8

When taking A and B chain and expressing them seperately in bacteria and they tried to purify the a and b chains they got a big insoluble mess as bacteria wasn’t able to fold chains properly.
Solution: tag insulin protein (subunit) of interest with a gene that produces a bacterial soluble protein (lac Z gene) thus helping solubise the insulin as well as the purification of subunits

We now have a bacterial promoter and a fusion protein of lac Z and insulin

• it is now transformed into ecology and we then purify that soluble fusion protein form the ecoli
• can now be transformed at put on antibiotic plate - select out single colonies and grow them up into a liquid culture
• but the purification of this will only get out enough for one injection, not sufficent
• pharmaceutical company uses large vats to purify it out a and b subunits from the bacteria
• everyday siphon off some of the bacteria and replace the bacteria with new stock and new media and start again

Question 9

Exaltation and purification process of insulin

Answer 9

1. Extract and purify LAC Z / insulin fusion proteins
2. Treat with cyanogen bromide to cleave A and B chains
3. Purify, mix A and B chains to form functional insulin (using promatography steps, separation by molecular weight or charge, depending on protein)

• then put in an environment that allows disulfide bonds to reform - exact same structure as if it was produced in ur own body - is also human so dont have complications of cow or pig insulin

Question 10

Advantages of prokaryotic systems

Answer 10

• relatively low cost
• high yield
• pathogen fee

Question 11

Disadvantages of prokaryotic systems

Answer 11

• proteins often partially folded
• inability to perform post-translational modifications

Question 12

Why would you make recombinant insulin in ammmalian cells and why wouldn’t you

Answer 12

• protein can be produces as a pre-pro-protein and processed efficiently
• will be secreted from cells
  
  • easier purification
• but more expensive to produce

Question 13

How to make recominnt insulin in eukaryotic cells - what’s different

Answer 13

Step 1: isolate cDNA for insulin (full length - signalBCA cDNA) - amplify by restiction enzymes by PCR
Step 2: clone into eukaryotic expression plasmid
Step 3: transform bacteria to produce more plasmid DNA (bacteria wont express becuase our promoter is mammalian - bacteria is just used as a way of generating more plasmid) and then transfect eukaryotic cells
Step 4: exract recombinant insulin from cell media
Step 5: purify insulin (using chromatography)

Question 14

How to decide what system to use

Answer 14

Question 15

Therapeutic proteins - recombinant human proteins - what does it mean and what does it require

Answer 15

• some proteins are only active when post-trasniflionally modified
• glycosylation - requires mammalian cells e.g erythropoietin = EPO

Question 16

EPO as a performance enhancing - blood doping - how does it work

Answer 16

• EPO increases RBC production
• increase in RBC leads to an increase in the oxygenation of muscle
• muscle uses oxygen to burn sugar and fats to generate ATP
• ATP is required for muscle contraction

Question 17

What do we need to make recombinant EPO for?

Answer 17

• many disease states result in lowered RBC counts
• chronic renal failure can cause a decrease in EPO levels, leading to anemia (decrease in RBC levels)
• cancer treatments (chemotherapy) may lead to anaemia
• administration of recombinant human EPO can restore RBC levels

Question 18

Structure of EPO

Answer 18

4 helix bundle

Question 19

When was EPO cloned

Answer 19

• in early 1980s

Question 20

EPO protein is ____-__________ modified

Answer 20

EPO is post-translationally modified (glycostlation)
- addition of carbohydrates to asparagine, serine or threonine residues

Question 21

In terms of EPO what is glycosylation

Answer 21

• addition of carbohydrates to asparagine, serine or threonine residues

Question 22

What is glycosylation important for

Answer 22

Biological function

Question 23

Where is EPO made

Answer 23

• in Chinese hamster ovary CHO cells

Question 24

Expression vector for EPO

Answer 24

Promoter - for transcription in mammalian cells (as we want to produce it in the Chinese hamster ovary cells)

• obtain human kidney RNA and reverse tracribe into cDNA - PCR with restriction sites on the end and then cut and paste it into the plasmid
• transformed into bacteria to make lots of copies
• after purifying it is then transfected into CHO cells, use big vast systems to grow them up in large amounts
• protein is secreted
• purified using chromatography

Question 25

What is pharming? What is an example?

Answer 25

- cells in culture cannot perform all post - translational modifications equally well e.g Y-carboxylation of glutamate - Y-carboxylation of certain glutamate residues is a feature of many proteins (including AT) involved in blood clotting - in 2006 (Europe) the first recombinant protein transgenic animal was approved as a drug - anti-thrombin (AT) - inhibits clotting factors - regulates clotting

Question 26

Details about AT

Answer 26

- AT deficiency may be hereditary (HD) or acquired - frequency of 1 in 2000 - 5000 - increased risk of inappropriate blood clotting - deficiency individuals receive AT when undergoing surgery or giving birth) - AT protein expressed in the milk of transgenic goats at lactation - AT then purified from other milk proteins

Question 27

What are recombinant DNA technologies important for?

Answer 27

The production of therapeutic proteins - they provide a safe means of getting human proteins for clinical use

Question 28

Are there are variety of cell systems available for producing recombinant proteins

Answer 28

Yes

Question 29

Are there a limited amount of cell systems available for producing recombinant proteins

Answer 29

No

Question 30

What systems must be used for proteins that require post-translational modification for their activity

Answer 30

Eukaryotic systems