Lecture 20 Flashcards
What are the key steps in producing recombinant proteins?
Isolate gene of interest, clone into expression vector, transform into bacteria for expression or isolate of more DNA for use in another expression system, grow cells expression protein of interest in appropriate system, isolate and purify protein
Why is insulin expression different?
Because it contains two chains (A and B) which are connected by disulfide bonds and it is also processed in the golgi
What is the solution to expression insulin in bacterial cells?
Express the A and B chain separately
When do the A and B chains get combined to form insulin?
In the purifying step
What are the advantages of using a prokaryotic system
Relatively low cost, receive a high yield and pathogen-free sample
What are the disadvantages of using a prokaryotic system
Proteins often partially fold if folding is complicated, bacterial cells are incapable of performing stable post-translational modifications
What are possible other systems to use when prokaryote systems are not sufficient?
Mammalian or transgenic animal systems
What makes mammalian systems more efficient?
Proteins can be produced as a pre-pro-protein and processed efficiently, the protein would be secreted from cells making purification easier
What is the disadvantage of mammalian systems?
They are very expensive and much slower
What would be different in making recombinant insulin in eukaryotic cells
Isolate cDNA for insulin, clone into eukaryotic expression vector, transform bacteria to produce more vector DNA, transfect eukaryotic cells, extract recombinant insulin from cell media, purify insulin
What is erythropoietin (EPO)?
A hormone that your kidneys naturally make to stimulate the production of red blood cells
Why must rEPO be expressed in a mammalian system?
Because it is post-translationally modified through glycosylation
What is glycosylation?
The addition of carbohydrates
What mammalian system is rEPO expressed in?
Chinese hamster ovary cells (CHO)
Why would we want to make rEPO?
Many disease states result in lowered red blood cell counts, leading to anaemia
What diseases can cause anaemia?
Chronic renal failure and cancer treatments (chemo)
What does EPO do?
Signals red bone marrow to make more red blood cells
Why is the glycosylation of EPO necessary?
Increases stability in the bloodstream, helps protein solubilise, ensures appropriate immune recognition and is required for proper biological functioning
How does rEPO enhance exercise performance?
Increases RBC leading to increased oxygenation of the muscle and the generation of more ATP
What is the composition of natural EPO?
It has a glycosylation pattern that is very specific to human cells, 4x sugar groups
How does rEPO differ from natural EPO?
The CHO cells add sugars in a manner that can differ significantly from human cells
How is rEPO detected?
Using isoelectric focusing, separating proteins based on their isoelectric point on a gel with a pH gradient. Proteins stop moving once they reached their pl point.
How do the isoelectric points of hEPO and rhEPO differ?
They have different charges due to the different glycosylation patterns, stopping at different pl points
What is pharming?
A biotechnology process that involves genetically modifying plants or animals to produce pharmaceutical substances
