Modern methods of producing medicinal products

Question 1

What are benefits of using recombinant products?

Answer 1

they are safer when producing vaccines for example its better to use a piece of the virus than the attenuated version of the virus and this means that production can be done on a large scale

Question 2

what is the large scale production system?

Answer 2

this uses huge 300L production vessels. and batch and continuous culture techniques. the fermenter uses an impellor which is for mixing and oxygenation. a fermenter train has to be used this is when you use a serious of increasing culture sizes to reach a good optical density to inoculate your culture into the ferment er. the trains are all going at the same time so that production doesnt have to stop

Question 3

what are the parts of the vector that is used to make bacterial recombinant products?

Answer 3

selectable marker
ORI
a multiple cloning site  which then will hold the DNA to be transcribed
the ribosomal binding site 
the promotor 
transcription terminator

Question 4

what needs to be a feature of the promotr?

Answer 4

it needs to be a strong promotor that you can control

Question 5

what is the transcription terminator for?

Answer 5

for if the promotor is particularly strong

Question 6

why is it good to control the promotor?

Answer 6

turn the gene off while the culture grows
then when the culture is dense
turn the gene on so that the toxic product is produced in a high quantity before the culture dies

Question 7

what is the problem with the lac promotor?

Answer 7

it is leaky even when the promotor is repressed

Question 8

what does the arabinose promotor need to be on?

Answer 8

araC - act - arabinose

and no glucose signalled by CRP-cAMP complex

Question 9

how does araC prevent trans when no arabinose?

Answer 9

binds to the DNA differently by binding to one of its previous binding sites and DNA downstream to cause bending of the DNA. meaning that there is no way that the RNA polymerise can bind - strongly off

Question 10

when may you go for a low copy number plasmid tightly controlled too?

Answer 10

when the product of the plasmid is toxic to the cell

Question 11

what can help companies stop spending loads on antiBs to make sure the plasmid is still in the cells?

Answer 11

using a low copy number plasmid as this is more likely to be retained in the cell as it is more stable.

Question 12

what does no enough HGH cause?

Answer 12

reduced bone growth, loss of muscle, increased fat this is nomrally made in the PG

Question 13

what is there a risk of getting HGH from the pituiatary?

Answer 13

prion disease

Question 14

what might have to be done to make a sequece usable in ecoli

Answer 14

chaning the dna sequene but still gettin gthe same aa sequence

Question 15

what prmotor is used to make HGH?

Answer 15

the tryptophan promotor which is only on when tryp isnt present

Question 16

what is the structure of the insulin protein translation initiatlly?

Answer 16

its made of the leader seuqence the sequence for one part of the pp and then a cleaved sequene then the other part of hte pp this will eventually be joined by a disulfide bond done when the chain folds together

Question 17

how is insulin made in e.coli?

Answer 17

two plasmid one for each bit using the lac promotor and then creating the right condition for the disulfide bond

Question 18

whars the yeast method to make insulin?

Answer 18

clone the whole gene in

in the yeast the protein folds spontaneously and forms the DS bonds and proteolytic cleavage also happens spontaneously

Question 19

what are the advntgaes of using yeast to make insulin?

Answer 19

• better for protein processing, like having the glycosylate products

Question 20

what yeast are used

Answer 20

pichia pastoris

sacc. cere

Question 21

where is glycosylation added?

Answer 21

to asparagine residues at the ER and Golgi

Question 22

what does glycosylation effect?

Answer 22

protein folding
immune recognition
clearane in the body

Question 23

what is the shuttle vector like?

Answer 23

ori and 2u(yeast)

ab marker and ura3 (yeast marker)

Question 24

what do you use instread of an antibiotic marker in yeast?

Answer 24

ura3 - allows for uracil porduction so you can kill the yeast that cant make this

Question 25

what is the gal1 promotor?

Answer 25

a promotor that increased expression 1000x fold in the presence ofgalactose, repressed by glucose - good for use in yeast

Question 26

whats the aox 1 promtoor?

Answer 26

induced by methanol, when on 30% of the cell protein will be the product from this promotor

Question 27

how is the hep b vaccine made?

Answer 27

its a subunit vaccine HbSAg vaccine mae in yeast because in e.coli it does not adopt its natural conformation

Question 28

what are the advnatgss of subunit vccines?

Answer 28

cheaper to make the antigen in large quantities these vaccines are also safer to make as no acual viriuses are used. and this is good when some biruses like hep b cant be made in culutrue

Question 29

what is hpv vaccine made of?

Answer 29

capsid proteins that are added in with sturctural paritcuales of the hpv so that the antigens are presented correctly - a viral mimic

Question 30

how is gardasil made?

Answer 30

in yeast made of the L1 protein

Question 31

how is cervarix made?

Answer 31

in insect culutre

Question 32

what does making the vaccine in this way mean for the stregnth fo the baccine?

Answer 32

better as the vaccine conc is high in the blood

Question 33

what needs to be done to antibodies to make them effective?

Answer 33

they need to be glycosylated

Question 34

what is the problem with using pichia to make monoclonal ABS?

Answer 34

they dont have the sam glycos system as us theres is more simple they have less of a varied sugar compoisiton and only glycos are ser and threonine

Question 35

what can we done to make pichia glycos like us?

Answer 35

engineer our glycos system into pichia so that the proteins made by it are like the ones in mammalian cells

Question 36

why is it better to use non mammalian cell lined?

Answer 36

cheaper
harder to grow them in large qunatity
greatr control over the cell line
and you can genetically manipulate these cell lines easily

Question 37

how can we make interfreron?

Answer 37

using e.coli its not glycosylated like by mammalian cells but works

Question 38

how caan we make osp A borelia vaccine?

Answer 38

using ecoli a good antibiral and good anti cancer as its anti proiferative

Question 39

what can glucagon made by yeast be used for?

Answer 39

hypoglycaemia and mnuscle relaxant