Lecture 20 - Recombinant proteins Flashcards
Key steps in producing a recombinant protein
- Isolate gene of interest
- Clone into expression plasmid - which one?
- Tranform into bacteria for expression or isolation of more DNA for use in another expression system
- Grow cells expressing protein of interest
- Isolate and purify the protein
Insulin formation process
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
Why can recombinant insulin protein easily be produced by bacteria?
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
Advantages of prokaryotic systems
Relatively low cost
High yield
Pathogen free
Disadvantages of prokaryotic systems
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
Making recombinant insulin in mammalian cells
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.
Making recombinant insulin in eukaryotic cells
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
Speed - best to worst
Bacteria, yeast, insect, mammalian, plants, transgenics
Cost - best to worst
Bacteria, yeast, insect, plants, mammalian, transgenics
Glycosylation - best to worst
Mammalian, transgenics, insect, plants, yeast, bacteria
Folding - best to worst
Mammalian, transgenics, insect, plants, yeast, bacteria
Therapeutic proteins - recombinant human proteins
Some proteins are only active when post-translationally modified
Glycosylation – requires mammalian cells e.g. erythropoietin = EPO
EPO
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
Why would we want to make recombinant EPO?
- 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
Expression vector for EPO
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.
Recombinant EPO
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
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?
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
Pharming - using whole animals to make recombinant proteins
- 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.
What advantages does a whole animal system have over a cell culture system in the generation of recombinant proteins?
post translational modifications can occur
Transfection
Move DNA (plasmid with eukaryotic promoter) into eukaryotic cell using non-viral methods
Recombinant insulin
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
