Expression Hosts Flashcards
What is an expression host?
When producing a protein product you need to use an organism to produce it. This is the host organism that it is expressed in.
What does the choice of expression host depend on?
#what the protein will be used for: -food and drug related have high levels of regulation -chemicals are less regulated #What type of protein is being produced -secreted -membrane -PTMs
What features does a good host have?
Allows the insertion or integration of a heterologous gene
Produces high quantities of the product
Produces the product correctly e.g folded, glycosylation
What features do proteins that are difficult to produce have?
Membrane proteins, PTMs and proteins that are prone to misfolding are difficult to produce
Name 8 expression host types.
Bacteria Insect cells Yeast Plants Mammalian cells Transgenic crops/animals Hybridoma Filamentous fungi
Describe general bacterial hosts
Takes about a week to evaluate.
Pros: quick growth, low costs, high yields, easy to manipulate
Cons: can’t make complex proteins e.g. PTMs
Describe general yeast hosts
Takes about a month to evaluate.
Pros: productivity and cost is similar to bacteria. High cell densities
Cons: processing is similar to mammalian cells
Describe insect cells as hosts
Takes about 2 weeks to evaluate. It uses bacmids and can make complex PTMs.
Pros: High yield, easy to manipulate, proteins tend to fold correctly, stress-resistance
Cons: Media is expensive, glycosylation is incorrect
Describe plants as hosts
Produces a yield of up to 25% dry cell weight. Used in biofuels e.g. green algae or crops
Pros: cheap media, can produce PTMs, folds correctly, good growth rate, good transformation, GRAS, good development time, reliable, scalable, carbon-neutral, robust, non-pathogenic.
Cons: Not much research into it, needs lots of space.
Describe mammalian cell hosts
Takes about 3 months to evaluate. CHO is a popular choice as there’s a genomic sequence, producing 1-5g/L.
Pros: Glycosylation is correct or very similar, there’s a reduction in the chance of rejection, PTMs happen.
Cons: costs a lot, complex groth requirements, takes a long time, yield is low, cellular stress can occur which leads to degredation, high genetic variability.
Describe transgenic hosts
Can use crops or animals produces 0.2% dry cell weight but the organisms are larger than cells.
Pros: Reduced risk of contamination, can use as a suplement, low cost, PTMs are made.
Cons:Needs lots of space, pesticides are used, takes a long time, toxic metabolites may be produced in plants.
Describe hybridomas as hosts
This fuses a tumour cell and a B cell together so that antibodies can be mass produced.
Pros: Good technology and idea
Cons: Low yield in bioreactors, doesn’t work on large scale.
Describe filamentous fungi as hosts
Good for producing enzyems e.g. amylases, lyases, xylanases, proteases, phytases. A.niger and A. oryzae have genomic sequences.
Pros: High yields of native proteins, PTMs are produced.
Cons: Low yields of recombinant proteins, fungal proteases cause degredation, unfolding occurs and there is a bottleneck in transcription.
Describe E.coli as a host
Gram -negative host that in 2009 had produced ~30% of the therapeutic proteins.
Pros: Well-known, well-characterised, quick to evaluate, mass produced and cost effective.
Cons: Not GRAS, no PTMs, produces endotoxins, uses inclusion bodies.
Describe Bacillus subtilis as a host
Gram-positive rod shape produces around 3g/L.
Pros: no lipopolysaccharides (endotoxins), no inclusion bodies, high levels of secretion, GRAS.
Cons: Bottlenecks in the secretory pathway, high degredation levels due to proteases, often incorrectly folded, poor targeting to the translocase, few suitable expression vectors, plasmids are fairly unstable, not that much knowledge, recombinagenic (integration doesn’t increase stability)
Describe Pichia pastoris as a host
Current popular choice, uses methanol as a carbon source.
Pros: High yields due to high cell densities, inducible/ tightly controlled promote AOX1, purification is fairly simple, methanol is cost effective, it doesn’t do extensive glycosylation.
Cons: Low cell specific activity, methanol is a safety hazard, secretory pathway bottlenecks, glycosylation pattern is different, cell gets stressed when it isn’t secreted so its then degraded.
Describe Sacchromyces cerevisae as a host
In 2009, 18.5% of recombinant pharmaceuticals were produced from S.cerevisae, e.g. hormones, vaccines, virus-like particles. There’s a genome database and it produces 9g/L.
Pros: Tends to be successful if bacteria wasn’t for mammalian proteins, GRAS, well known, there’s a library of vectors and it’s low cost.
Cons: waste products (ethanol) from fermentation limit the cell densities, hyperglycosylation occurs, N-glycans are allergenic.
Describe virus-like particles
These are like viruses but contain no viral genetic material. They are fairly low yield but have a huge variety. They are used in vaccines and in transgenic plants as you can eat it and become immune.
Describe cell-free protein synthesis and 4 examples of lysates
This uses lysates of cell machinery which has no dependence on cell growth.
e.g. E.coli, rabbit reticulocyte, wheat germ extract and insect cells
What would you consider when choosing a host to produce single chain variable fragments (ScFv)?
These can be used as therapeutic drugs so you need to consider:
- Uses
- Secretion levels
- Time frames
- Expression levels
- PTMs
Describe the case study of Pichia and how it was further improved
Pichia was shown to not hyper-mannosylate which reduces rejections, and have an optimal feeding stategy so 1.6g/L of monclonal antibodies in 1200L can be produced.
Improvements:
-Increase the titre; increase gene dosage, change promoters, gene fusions
-Glycosylation patterns; adding and deleting various genes so they are spatial and temporally expressed for glycosylation.
-Reduce secretory pathway bottlenecks; co-expression folding chaperones, specialised strains, codon optimisation, modified feeding strategies or alternative promoters.
Describe the pros & cons of E.coli lysate
Pros:simple, cost effective, high yield, quick synthesis, easy to make yourself.
Cons: limited PTMs, expensive to buy.
Describe the pros & cons of Wheat germ extract lysate
Pros: high-throughput and gives a high yield for complex proteins
Cons: low yield of extract, poor modification tools, takes a while and is complex to prep
Describe the pros & cons of Rabbit reticulocyte lysate
Pros: quick prep, does PTMs
Cons: you need a licence to use animal tissues, low protein yield, lots of endigenous proteins
Describe the pros & cons of insect cell lysate
Pros: quick prep, PTMs, signal sequence processing
Cons: cell cultivation is costly and slow.
