Lecture 20 - Recombinant proteins

Question 1

Key steps in producing a recombinant protein

Answer 1

1. Isolate gene of interest
2. Clone into expression plasmid - which one?
3. Tranform into bacteria for expression 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

Insulin formation process

Answer 2

Have an mRNA strand and use reverse transcriptase in order to create a cDNA which has no introns - they have been cleaved out as the bacteria can’t splice out the introns.

Insulin is produced in the pancreas as a pre-proprotein that is further processed by Golgi
– This won’t happen in bacteria
Solution = express chain A and B separately in bacteria

When they are extracted and purified…. A and B chains are mixed together in a way that allows the formation of disulphide bonds and this produces the active insulin, looks nearly identical to the ones produced in the pancreas

Question 3

Why can recombinant insulin protein easily be produced by bacteria?

Answer 3

Recombinant DNA is a technology scientists developed that made it possible to insert a human gene into the genetic material of a common bacterium. This “recombinant” micro-organism could now produce the protein encoded by the human gene. There, the recombinant bacteria use the gene to begin producing human insulin

Question 4

Advantages of prokaryotic systems

Answer 4

Relatively low cost
High yield
Pathogen free

Question 5

Disadvantages of prokaryotic systems

Answer 5

Proteins often partially folded/dont fold properly - for example with insulin you are making the two subunits independently and doing post processing to form a fully functional mature protein
Inability to perform post-translational modifications

Question 6

Making recombinant insulin in mammalian cells

Answer 6

Why?
– Protein can be produced as a pre-pro-protein and processed efficiently 
– Will be secreted from cells – easier purification 
– BUT more expensive to produce.

Question 7

Making recombinant insulin in eukaryotic cells

Answer 7

Step 1. Isolate cDNA for insulin 
Step 2. Clone into eukaryotic expression plasmid 
Step 3. Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells 
Step 4. Extract recombinant insulin from cell media 
Step 5. Purify insulin

Question 8

Speed - best to worst

Answer 8

Bacteria, yeast, insect, mammalian, plants, transgenics

Question 9

Cost - best to worst

Answer 9

Bacteria, yeast, insect, plants, mammalian, transgenics

Question 10

Glycosylation - best to worst

Answer 10

Mammalian, transgenics, insect, plants, yeast, bacteria

Question 11

Folding - best to worst

Answer 11

Mammalian, transgenics, insect, plants, yeast, bacteria

Question 12

Therapeutic proteins - recombinant human proteins

Answer 12

Some proteins are only active when post-translationally modified
Glycosylation – requires mammalian cells e.g. erythropoietin = EPO

Question 13

EPO

Answer 13

EPO as a performance enhancing drug – blood doping

• increase in RBCs leads to an increase in the oxygenation of muscle
• muscle uses oxygen to burn sugar and fats to generate ATP
• ATP required for muscle contraction
• gene cloned in early 1980s
• protein is post-translationally modified (glycosylation Addition of carbohydrates to asparagine, serine or threonine residues
• glycosylation important for biological function
• made in Chinese Hamster Ovary (CHO) cells

Question 14

Why would we want to make recombinant EPO?

Answer 14

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

Question 15

Expression vector for EPO

Answer 15

Promoter for transcription in mammalian cells
Human EPO gene
Antibiotic resistance gene that is used after transformation of the bacteria to produce multiple copies of the plasmid.

Plasmid is placed into CHO cells and then protein purification must occur to get the functioning protein.

Question 16

Recombinant EPO

Answer 16

EPO stimulates RBC formation and Hb production
Human EPO is extensively and uniquely glycosylated which is a type of post translational modification and this is essential to be done for protein function therefore done in eukaryotic cell host.
Made in Chinese hamster ovary cells (CHO cells)

Eukaryotic expression vector includes … 
Eukaryotic host cell promoter (e.g. CHO promoter) to which eukaryotic host RNA polymerase binds to initiate transcription of human gene - promoter must be able to bind the eukaryotic transcription factors that are produced by the cell therefore must be a eukaryotic promoter sequence. Promoter must match your host cell because it is the host cell that will produce the transcription factors that will bind to the promoter that will recruit the RNA polymerase allowing transcription to occur. 
Target gene e.g. human EPO gene 
ORI 
Antibiotic resistance gene

Question 17

If EPO was generated in a bacterial system, would the recombinant protein be active in the human body of patients that require an EPO therapy?

Answer 17

No, protein is post-translationally modified (glycosylation Addition of carbohydrates to asparagine, serine or threonine residues)
glycosylation important for biological function
- made in Chinese Hamster Ovary (CHO) cells

Question 18

Pharming - using whole animals to make recombinant proteins

Answer 18

• cells in culture cannot perform all post-translational modifications equally well e.g. γ-carboxylation of glutamate
• γ-carboxylation of certain glutamate residues is a feature of many proteins involved in blood clotting
• in 2006 (Europe) the first recombinant protein produced from a transgenic animal was approved as a drug – anti-thrombin (AT)

-AT deficiency may be hereditary or acquired
-frequency of 1 in 2000-5000
-increased risk of inappropriate blood clotting
-deficient individuals receive AT when undergoing surgery or giving birth
AT protein expressed in the milk of transgenic goats at lactation
• AT then purified from other milk proteins
There is a milk specific promotor which responds to hormonal signals that induce lactation, there is also a human AT gene in the plasmid.

Question 19

What advantages does a whole animal system have over a cell culture system in the generation of recombinant proteins?

Answer 19

post translational modifications can occur

Question 20

Transfection

Answer 20

Move DNA (plasmid with eukaryotic promoter) into eukaryotic cell using non-viral methods

Question 21

Recombinant insulin

Answer 21

Human insulin has 2 short polypeptide chains that are linked together by disulphide bonds. It is a small simple protein with no post translational modification required therefore can use a prokaryotic host cell.

Bacterial expression vector will include …
Bacterial host cell promoter sequence to which bacterial host RNA polymerase binds to initiate transcription of a human gene
Target gene (e.g. human insulin chain A or chain B)
Ori - to ensure that the cell makes many copies of this plasmid
Antibiotic resistance gene