lecture 20

Question 1

what are the five rough key steps in making a recombinant protein

Answer 1

isolate the gene of interest
clone into expression plasmid
transform into bacteria for expression or isolation
grow cells expressing protein of interest
isolate and purify the protein

Question 2

why cant a full human insulin molecule be produced by bacteria

Answer 2

Insulin is produced in the pancreas as a pre-proprotein that is further processed by golgi. making whole insulin therefore wont work as bacteria lack the golgi body.
prokaryotes also don’t have the needed proteases to cleave the C chain

Question 3

whats the solution for not being able to produce insulin in individual bacteria

Answer 3

produce the chain A in one bacteria and chain B in another

Question 4

advantages of prokaryotic systems for producing protein

Answer 4

low cost, high yield and free of pathogens

Question 5

disadvantages of protein production in prokaryotic systems

Answer 5

proteins often partially fold in prokaryotes
prokaryotes are unable to perform post-translational modifications

Question 6

2 advantages and 1 disadvantage of producing recombinant insulin in mammal cells

Answer 6

protein can be produced as a pre=pro-protein and processed efficiently.
will be secreted from cells making easier purification
but its more expenny

Question 6

5 steps for making recombinant genes in eukaryotic cells

Answer 6

isolate the cDNA.
clone into eukaryotic expression plasmid
transform bacteria with the plasmid to make more of the plasmid then transfect the plasmids into eukaryotes extract recombinant insulin from cell media
purify the insulin

Question 6

4 factors to account for when chosing cell type for transgenes

Answer 6

speed, cost, can it do PTM? folding

Question 7

some proteins are only active when post-translationally modified, example

Answer 7

erythropoietin

Question 8

what PTM are done to EPO

Answer 8

protein is PTM by glycosylation. the addition of carbohydrates to asp, ser and threonine residues

Question 9

whats pharming

Answer 9

using animals to make recombinant proteins

Question 9

what is EPO produced in

Answer 9

Chinese hamster ovaries

Question 9

whats the antithrombin gene do

Answer 9

helps in blood clotting

Question 9

how is AT gene produced in the transgenic animals

Answer 9

we dont want it produced in every cell as that would affect the animal. thus we place a milk promoter on AT gene. so that AT is only produced and excreted in milk,

Question 10

what processes do we use to get the plasmid into the cells

Answer 10

Uses either chemical transfromation or electrophoration.

Question 11

in premature insulin, the A and B chains are held by C chain, what cleaves C chain

Answer 11

proteases

Question 12

when producing the A and B strands in bacteria separately, the insulin is insoluble. how does one get around this

Answer 12

Thus the protein was tacked with a soluble protein called Lac-z. Lac-z helps solubise the insulin. These genes are fused to the A and B chains.