How to create recombinace

Question 1

What are the steps to creating a recombinant protein

Answer 1

1. isolate gene of interest
2. Clone DNA into exspression plasmid -> vector
3. transform bacteria with plasmid
4. grow cell in appropriate exspression system
   - dependant on plasmid promotor - could be bacteria, mamalian, transgenic animal
5. extract and purify protein
   - easy in eukarotic cells = excreation

Question 2

What is the challage of growing insulin

Answer 2

Insulin DNA is preproinsulin -> signal protein + A B and C chains
folding drops C and signal proteins leaving mature insulin with A and B chains

Question 3

How do we grow insulin in bacteria

Answer 3

ISolate A and B chains seperate
create 2 plasmids
replicatie + exspress in 2 seperate bacteria
extract chains -> oxidise to enable functional insulin to form

Question 4

what are the pros and cons of prokaryotic cells, eukaryotic cells and transgenic animals

Answer 4

Prokarotes:
pro: Cheap, fast high yeild,
cons: Cant PTM or complex fold

Eukaryotes:
pros: can undergo PTM
cons: slow + exspensive

Transgenic animals:
pro: can do gamma-carboxylation,
disadvantage: slow expensive, challanging

Question 4

What is EPO and why and how do we synthesise it

Answer 4

EPO is a 4 helix bundel which increase RBC count
requires 4 glycosylations in PTM
- solubility, function, immune recognition, stability

used to help conditions with anemia
- renal kidney failure, cancer varients

done in chineese hamster ovary cells as they undergo glycosylation -> not exactly same as hEPO due to different glycosylation patterns

Question 5

How is insulin process different in eularotic cell why is this uncommon

Answer 5

Eukarote can undergo PTM and complex folding
Uses whole DNA with signal protein ABC chains

Eukaryotic promotor in vector
transfect eukarotic cell after replication in bacteria
Insulin secreated from cells

Uncommon -> takes a longer time, lower yeild, more exspensive

Question 6

how is antithrombin farmed and why

Answer 6

Antithrombin is a blood thinner -> used in birth + surgery for people with deficeny

farmed in transgenic animasl due to complex PTM
requires gamma-carboxylation -> required animal

process: goat mammory gland DNA mixed with AT gene -> put into oocyte -> grows into goat -> milk produced has AT protein -> purified for use

Question 6

What is a problem with rhEPO and how is this detected

Answer 6

Blood doping = using rhEPO to boost RBC and performance

isoelectric focusing -
tecnique used to speperate proteined based on isoelectric point using electric feilds
Result is visualised with antiEPO antibodys -> rhEPO and hEPO show differently