Molecular Genetics & Biotechnology - Lecture Twenty Flashcards
Using Recombinant DNA Technologies to Make Proteins
Steps to producing a recombinant protein
Isolate gene of interest (separate A and B chains)
Clone into expression plasmid
Transform 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
Step One - How do we remove the two introns and two exons from insulin?
By isolating the mRNA from the pancreas, reverse transcribe that RNA
Insulin
Produced in the pancreases as a pre-pro-protein that is further processed by Golgi
Advantages of prokaryotic systems
Relatively low cost
High yield
Pathogen free
Disadvantages of prokaryotic systems
Proteins often partially folded
Inability to perform post-translational modification
Why make recombinant insulin in mammalian cells?
Protein can be produced as pre-pro-protein and processed efficiently
Will be secreted from cells which is easier purification
Limitations of making recombinant insulin in mammalian cells?
More expensive to produce
Steps to making recombinant insulin in eukaryotic cells
Isolate cDNA for insulin
Clone into eukaryotic expression plasmid
Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells
Extract recombinant insulin from cell media
Purify insulin
Glycoslation
Requires mammalian cells
Blood doping
Increases red blood cells which leads to an increase in the oxygenation of muscles, which burn sugar and fats to generate ATP. ATP is required for muscle contraction
Why would we want to make a recombinant for erythropoietin (EPO)
Many disease states result in lowered red blood cell counts, administration of recombinant human EPO can restore red blood cell levels
Chronic renal failure
Can cause a decrease in EPO levels, leading to anaemia
Cancer treatments (chemotherapy)
May lead to anaemia
Anti-thrombin (AT)
Blocks enzymes in intrinsic pathway which leads to blood clotting
