Lecture 20 - recombinant proteins

Question 1

outline the steps required to make recombinant proteins

Answer 1

1. isolate the gene of interest
2. clone into expression plasmid (which one)
3. transform into bacteria for expressions or isolation of more DNA for use in another expression system
4. grow cells expressing protein of interest
5. isolate and purify the protein

Question 2

what is special about cDNA

Answer 2

it does not contain introns

Question 3

using insulin as an example why must the C chain be removed

Answer 3

because when the eukaryotic gene is expressed in bacteria, bacteria do not have the mechanism to remove the intron

Question 4

what is an intron

Answer 4

noncoding sections of an RNA transcript, or the DNA encoding it, that are spliced out before the RNA molecule is translated into a protein

Question 5

using insulin as an example what is added to the plasmid that is fused to the insulin gene subunit and what does it do

Answer 5

lac Z gene is fused and it stops misfolding of A and B chains

Question 6

why is lac Z removed at the end of the steps to making recombinant protein (inuslin)

Answer 6

because we do not want lac Z to be part of the mature protein

Question 7

what are the advantages of prokaryotic systems

Answer 7

• relatively low costs
• heigh yield
• pathogen free

Question 8

what are the disadvantages of prokaryotic systems

Answer 8

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

Question 9

is making recombinant insulin in mammalian cells cheaper or more expensive

Answer 9

more expensive

Question 10

what is different about using mammalian cells instead of prokaryotic systems for producing recombinant insulin

Answer 10

cDNA = fine to have the signal sequence as B-C-A as mammalian cells will be able to cleave C

Question 11

what is the process for making recombinant insulin in eukaryotic cells

Answer 11

1. isolate CDNA for insulin
2. clone into eukaryotic expression plasmid
3. transform bacteria to produce more plasmid DNA and transfect eukaryotic cells
4. extract recombinant insulin from cell media
5. purify insulin

Question 12

in terms of speed which is best used

Answer 12

bacteria

Question 13

what is transfection

Answer 13

introducing genetic material into eukaryotic cells

Question 14

in terms of cost which is best

Answer 14

bacteria

Question 15

why are mammalian cells used to produce EPO

Answer 15

because the protein is post translationally modified so can not be done in bacteria

Question 16

what is pharming

Answer 16

using whole animals to make recombinant proteins

Question 17

why are transgenic animals needed antithrombin (AT)

Answer 17

because cells in culture can not perform all post translational modifications equally well (e.g y-carboxylation)

Question 18

how does making recombinant proteins (atnithromnin) work in transgenic animals

Answer 18

they are transfected with plasmid as an embryo, hopefully it would’ve been taken up by the animal

• antithrombin then expressed in the milk of the animal
• then purified from other milk proteins