Lecture 2 Flashcards
What do protein drugs do?
Replace a protein that is abnormal or deficient in a particular disease or augment the body’s supply of a beneficial protein.
What is the process of mRNA display?
- Transcription of the dsDNA library
- Ligation of a puromycin molecule to the 3’ end of each mRNA
- Translation of the mRNA → at the end of the sequence, puromycin will enter the site at the stop codon and get incorporated into the peptide. This means that the protein and mRNA will be linked together by the puromycin
Purification of the products - The mRNA is reverse transcribed to yield a double stranded molecule with mRNA and cDNA
- The mRNA-protein library is exposed to the target protein
- The unbound protein is washed off and the bound mRNA protein is eluted.
- PCR is done to amplify the DNA that codes for the protein that binds well to the target protein.
What are the advantages of using E coli cells for cell engineering?
- Well characterised → facilitates genetic manipulation.
- Rapid expansion (doubling time = 30 min). Ease of scaling up.
- High expression levels of recombinant protein (100- 1000 mg/L).
- Grows rapidly on simple and inexpensive media. Low production cost.
What are the disadvantages of using E coli for cell engineering?
- Recombinant protein accumulates intracellularly.
- Form insoluble aggregates called inclusion bodies (aggregates of misfolded or unfolded proteins) because the protein is too large to be folded properly
- Lack the ability to perform post-translational modifications (e.g. glycosylation).
- Presence of lipopolysaccharides (LPS) on its surface that act as pyrogens – endotoxins
(The immune system can recognize the LPS so it has to be removed)
What are CHO cells and where are they derived from?
Chinese Hamster Ovary cells. They are the epithelial cells derived from the ovary of the Chinese hamster
What are the advantages of using CHO cells for cell engineering?
- They are capable of adapting and growing in suspension culture, so they are ideal for large scale culture.
- Less risk as few human viruses can propagate in them.
- Grow in serum-free and chemically defined media.
- Allow post-translational modifications
- Can be manipulated by genetic engineering techniques to produce higher yield of recombinant proteins.
What are the disadvantages of using CHO cells for cell engineering?
- Laborious construction of over-expressing cells.
- Complex and expensive media. High production cost
- Low growth rates (doubling time = 24h). Difficult scaleup.
- Lower levels of protein expression (1-100mg/L), and they also produce many other proteins for itself
- Batch to batch variation depending on culture conditions
How do you choose what host cell to use for cell engineering?
Choose eukaryotic host cells for:
1. Large proteins of >100kDa
2. When post translational modifications are essential
3. In proteins where solubility and naive folding is crucial
Choose prokaryotic host cells for:
1. Small proteins of <30kDa
2. When you want high yields at low cost
What are the challenges that come with producing biologics?
- They must be processed under tightly controlled conditions
- Need to be able to consistently produce safe, pure and potent product
- Prevent introduction of environmental and microbial contamination - Biologics are susceptible to changes in temperature and light
- They have a limited shelf-life
- They typically need refrigeration, are frozen during storage or preservatives.
What are some conditions that need to be controlled in a cell culture?
Temperature, CO2 level, O2 level, humidity
What are some issues that arise during cell culture?
- The cell cultures may get contaminated with bacteria, fungi and mycoplasma
- Cell characteristics may change after a long period of continuous growth due to mutation and genetic drift
- The physiology of cells may change dramatically in the event of nutrient depletion and waste byproduct accumulation
What is the device used to culture a large number of cells for protein production?
Bioreactor
True or false?
One advantage of protein drugs is that they can be genetically modified to have greater interaction with the substrate for a stronger response.
True.
What kind of bioreactor is used with microcarriers?
Stirred tank bioractors
How do cells stick to microcarriers?
Microcarriers are coated with a positively charged polymer to allow cells to stick to it.
