Expression Hosts

Question 1

What is an expression host?

Answer 1

When producing a protein product you need to use an organism to produce it. This is the host organism that it is expressed in.

Question 2

What does the choice of expression host depend on?

Answer 2

#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

Question 3

What features does a good host have?

Answer 3

Allows the insertion or integration of a heterologous gene
Produces high quantities of the product
Produces the product correctly e.g folded, glycosylation

Question 4

What features do proteins that are difficult to produce have?

Answer 4

Membrane proteins, PTMs and proteins that are prone to misfolding are difficult to produce

Question 5

Name 8 expression host types.

Answer 5

Bacteria
Insect cells
Yeast
Plants
Mammalian cells
Transgenic crops/animals
Hybridoma
Filamentous fungi

Question 6

Describe general bacterial hosts

Answer 6

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

Question 7

Describe general yeast hosts

Answer 7

Takes about a month to evaluate.
Pros: productivity and cost is similar to bacteria. High cell densities
Cons: processing is similar to mammalian cells

Question 8

Describe insect cells as hosts

Answer 8

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

Question 9

Describe plants as hosts

Answer 9

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.

Question 10

Describe mammalian cell hosts

Answer 10

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.

Question 11

Describe transgenic hosts

Answer 11

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.

Question 12

Describe hybridomas as hosts

Answer 12

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.

Question 13

Describe filamentous fungi as hosts

Answer 13

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.

Question 14

Describe E.coli as a host

Answer 14

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.

Question 15

Describe Bacillus subtilis as a host

Answer 15

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)

Question 16

Describe Pichia pastoris as a host

Answer 16

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.

Question 17

Describe Sacchromyces cerevisae as a host

Answer 17

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.

Question 18

Describe virus-like particles

Answer 18

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.

Question 19

Describe cell-free protein synthesis and 4 examples of lysates

Answer 19

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

Question 20

What would you consider when choosing a host to produce single chain variable fragments (ScFv)?

Answer 20

These can be used as therapeutic drugs so you need to consider:

• Uses
• Secretion levels
• Time frames
• Expression levels
• PTMs

Question 21

Describe the case study of Pichia and how it was further improved

Answer 21

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.

Question 22

Describe the pros & cons of E.coli lysate

Answer 22

Pros:simple, cost effective, high yield, quick synthesis, easy to make yourself.
Cons: limited PTMs, expensive to buy.

Question 23

Describe the pros & cons of Wheat germ extract lysate

Answer 23

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

Question 24

Describe the pros & cons of Rabbit reticulocyte lysate

Answer 24

Pros: quick prep, does PTMs
Cons: you need a licence to use animal tissues, low protein yield, lots of endigenous proteins

Question 25

Describe the pros & cons of insect cell lysate

Answer 25

Pros: quick prep, PTMs, signal sequence processing
Cons: cell cultivation is costly and slow.