BIOC192 Lecture 20 - Recombinant proteins or using bugs to treat diabetes

Question 1

what are the 5 key steps in producing a recombinant protein for insulin?

Answer 1

1) isolate gene of interest: insulin from mRNA from cDNA (beta cells from the pancreas) using reverse transcriptase
2) clone into expression plasmid: clone into a bacterial expression plasmid, requires restriction enzymes of ecoR1 and DNA ligase
3) transform into bacteria for expression or isolation of more DNA for use in another expression system: incubate plasmid with E.coli + antibiotic (bacteria with a antibiotic resistance gene are the only ones that survive)
4) grow cells expressing protein of interest (in large volumes resulting in expression insulin)
5) isolate and purify protein (eliminate contaminants)

Question 2

what is step 1 of recombinant insulin production?

Answer 2

• isolated pancreatic tissue and beta cells that are expressing the mRNA
• use reverse transcription to produce cDNA (copy DNA) from the mRNA
• C chain is spliced out
• pcr amplified

Question 3

why is it important that cDNA (copy DNA) has no introns?

Answer 3

because bacteria can not splice out introns

Question 4

what are the 3 chains of insulin?

Answer 4

1) A chain
2) B chain
3) C chain

Question 5

what is a pre-protein?

Answer 5

single sequence protein

Question 6

where is the pre-protein further processed?

Answer 6

insulin is produced in the pancreas as a pre-protein that is further processed by the golgi
-this does not occur in bacteria

Question 7

what is the relationship between the production of pre-protein and bacteria in terms of the chains?

Answer 7

bacteria express chain A and B seperately

Question 8

what is step 2-3 of recombinant insulin production?

Answer 8

clone gene into expression plasmid(s) and transform bacteria

• bacterial plasmid transformed into bacteria (2 separate hosts from the A chain and the B chain)\
• isolate the subunits of chain A and chain B

Question 9

what is step 4 of recombinant insulin production?

Answer 9

grow bacteria expressing insulin A and B chains

• selected on an antibiotic resistant agar plate
• screened the colonies. to distinguish between chains
• produce between 100mL-500mL of the insulin protein in the lab

Question 10

what is step 5 of recombinant insulin production?

Answer 10

extract and purify

• extracted an purified insulin fusion proteins
• treat with cyanogen bromide to cleave A and B chains
• purify, mix A and B chains to form functional insulin (disulphide bonds)

Question 11

what are the advantages of prokaryotic systems?

Answer 11

• relatively low cost
• high yield
• pathogen free

Question 12

what is the disadvantage of prokaryotic systems?

Answer 12

• proteins often partially folded

- inability to perform post-translational modifications

Question 13

what is the purpose of making recombinant insulin in mammalian cells?

Answer 13

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

Question 14

what is the difference in steps in producing recombinant insulin in eukaryotic cells (5 steps)?

Answer 14

1) isolate cDNA for insulin
2) Colin into eukaryotic expression plasmid
3) transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells
4) extract recombinant insulin from cell media
5) purify insulin

Question 15

what are 4 reasons to make EPO (erthyropoetin)?

Answer 15

1) many disease states result in lowered red blood cell counts
2) chronic renal failure can cause a decrease in EPO levels, leading to anaemia
3) cancer treatments (chemotherapy) may lead to anaemia
4) administration of recombinant human EPO can restore red blood cell levels