Lecture 20: Recombinant proteins Flashcards
Why can recombinant insulin protein easily be produced by bacteria?
As it can be processed fully in a bacterial system by expressing chain A and B separately in bacteria (No PTM)
Step 1 – Obtain human insulin cDNA
Step 2 - Clone gene into expression vectors
Step 3 -Transform bacteria
Step 4 - Grow bacteria expressing insulin A and B chains
Step 5. Extract and purify insulin
Bacteria are inexpensive to culture, making the production of recombinant insulin both scalable and cost-effective.
What are the advantages and disadvantages of using a prokaryotic system to generate human recombinant proteins?
Advantages:
* relatively low cost
* high yield
* pathogen free
Disadvantages:
* proteins often partially folded
* inability to perform stable post-translational modifications
What are the advantages and disadvantages of using a eukaryotic system to generate human recombinant proteins?
Advantages:
- Eukaryotic cells can add complex modifications (like sugars) needed for the protein to work properly.
- Proteins often work better and are more similar to human proteins.
- Can produce more complex proteins that bacteria can’t.
Disadvantages:
- More expensive to grow and maintain than bacteria.
- Grow slower and produce less protein.
- Higher risk of contamination.
- More complex and difficult to manipulate.
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, as the Protein is post-translationally modified (glycosylation)
Bacterial systems cannot perform these actions thus the EPO generated would not be the same and therefore not active in the human body
If an athlete has used rhEPO as a performance enhancer, how are we able to detect this?
Natural EPO
* Produced primarily in the kidneys and has a glycosylation
pattern that is specific to human cells.
rhEPO
* Produced in Chinese Hamster Ovary (CHO) cells. These
cells add sugars in a manner that can differ significantly
from human cells.
These differences in glycosylation can be detected using Isoelectric focusing (IEF). The Gel has a pH gradient. Proteins stop moving in an electric field once they reach their pI. hEPO and rhEPO will stop moving at different locations on the gel.
Thus we can detect performance enhancer
What advantages does a whole animal system (pharming) have over a cell culture system in the generation of recombinant proteins?
Complex Modifications:
Can produce proteins with even more complex post-translational modifications and structural features that are more similar to those in humans. e.g. y-carboxylation of glutamate (protein involved in blood clotting)
Natural Environment:
Provides a more natural biological context for protein folding and function.
However, it is more costly and ethically complex than cell culture systems.
What protein production system would be used to produce glycosylated proteins?
Best option is Mammalian cell culture/eukaryotic cells
Transgenic animals/pharming (less likely unless very complex) - more expensive
Describe a system that could be used to produce human EPO: Why this system?
Mammalian cell culture (eg. Chinese Hamster Ovary cells)
This system is needed as EPO requires post translational modification (glycosylation) that bacteria can’t do
What system would be used to produce a protein that requires y-carboxylation eg. Antithrombin?
Transgenic animals/Pharming.
This process is too complex for a simple cell culture thus requires an animal to provide the correct post translational modifications
