Lecture 20 - Using Recombinant DNA Technologies to Make Proteins Flashcards
The genetic code is____?
Universal
_____ reverse transcribes into ______ to generate a DNA sequence that doesn’t contain introns
mRNA, cDNA
What are the key steps in producing a recombinant protein? (5)
Isolate, Clone, Transform, Grow, Purify
- Isolate gene of interest
- Clone into expression vector
- Transform into bacteria for expression or isolation of more DNA for use in another expression system.
- Grow cells expressing protein of interest in appropriate expression system
- Isolate and purify protein
What is Preproinsulin processed into?
Proinsulin via signal peptide cleavage
What is Proinsulin process into?
Mature insulin via C-Peptide cleavage
What 2 chains are left in the mature insulin produced?
A and B chains
How are the A and B chains held together?
Via disulphide bonds
Where is insulin produced and where is it fuether processed?
produced in the pancreas, further produced in the golgi. (does not happen in bacteria)
What is the first step in recombinant insulin production?
Obtaining human insulin cDNA (via reverse transcription where intron are removed)
What is the second step in recombinant insulin production?
Close gene into expression vectors (two vectors are needed, one for A and B subunits)
What are needed within the recombinant vectors?
- Bacterial promoter
- Origin of replication
- Antibiotic resistance gene
What is the third step in recombinant insulin production?
Transform bacteria (into seperate colonies)
What is the fourth step in recombinant insulin production?
Grow bacteria expressing insulin A and B chains
What is the fifth step in recombinant insulin production?
Extraction and purification of viral vectors from sample.
What is the sixth and last step in recombinant insulin production?
Combining of A subunit and B subunit vectors via disulphide bonds.
What method was first used to create disulphide bonds?
Air oxidiation
Advantages of prokaryotic systems:
- relatively low cost
- high yield
- pathogen free
Disadvantages of prokaryotic systems:
- Proteins often partially folded
- inability to preform stable post-translational modifications.
What does recombinant insulin using mammalian cells allow?
- Protein can be produced as pre-pro-protein and processed efficiently
- The protein would be secreted from cells (easier purification)
- More expensive.
What would be the steps in making recombinant insulin in eukaryotic cells instead of bacterial?
- Isolate cDNA for insulin.
- clone into eukaryotic expression vector
- Transform bacteria to produce more vector DNA
- Transfect eukaryotic cells
- Extract recombinant insulin and purify
Whats the most appropriate system for Glycosylation and Folding?
Mammalian
What does EPO stand for?
Erythropoietin
What is the structure of EPO?
- 4 helix bundle (simple and easy to fold)
- Post translationally modified (glycosylation)
- Made in mammalian cells due to PTM
rEPO is made in what animal cells?
Chinese Hamster Ovary cells. (CHO)
Where is EPO produced?
The kidneys
What does EPO act to do?
Stimulate bone marrow to produce red blood cells.
(lack of EPO can lead to anemia)
What does glycosylation of EPO do to the structure?
- increases the stability in bloodstream
- Required for proper biological function
- Helps the protein solubilize in the blood stream
- Ensures appropriate immune recognition.
What types of cells are recombinant human EPO produced in?
Mammalian cells (due to need of PTM)
What is the process of expression of the rhEPO vector?
- Eukaryotic expression vector needed.
- Specific promoter for transcription in mammalian cells
- Purification and administration.
- Injected into bloodstream always.
How is EPO considered a performing enhancing drug?
Due to increase in RBCs, this leads to an increase in the oxygenation of muscle. More ATP is generated from oxygen in muscle burning sugar and fats.
Is rhEPO and EPO the same?
No, rhEPO is designed to mimic natural EPO, but has cells that add sugars in a manner that can differ significantly from humans.
What method can be used to test for rhEPO and EPO?
Isoelectric focusing. (IEF)
How does Isoelectric focusing work?
Separates proteins based on their isoelectric point.
Due to rhEPO and EPO have different isoelectric points, a visual difference will appear due to gel moving patterns.
What is pharming?
Using a whole animal to make recombinant proteins.
Do all cells in culture perform all PTMs equally?
No, an example being Gamma Carboxylation (PTM)
What was the first protein produced for a transgenic animal?
Antithrombin (AT)
What does Antithrombin do?
Acts as a natural blood thinner to prevent blood clotting.
Where is the AT protein expressed?
In the milk of transgenic goats at lactation
What is the process in reproducing the AT protein?
Promotor and AT are inserted into egg,
then into goat,
offspring produced.
