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Biotechnology > Expression Hosts > Flashcards

Expression Hosts Flashcards

1
Q

What percentage of biopharmacutical srae produced by Escherichia coli and Saccharomyces cerevisiae?

A

50%

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2
Q

What is a hybridoma?

A

An indefinitely reproducing cell that produces a specific antibody
(Produced by injecting a specific antigen into a mouse, collecting an antibody-producing cell from spleen, and fusing it with a tumor cell.)

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3
Q

What was the first recombinant protein produced?

A

Humulin

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4
Q

What expression host produced the first recombinant protein?

A

E.coli

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5
Q

What is an endotoxin?

A

Lipopolysaccharides -
large molecules consisting of a lipid and a polysaccharide
- found in the outer membrane of Gram-negative bacteria

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6
Q

What is a GRAS organism?

A

‘Generally Recognised As Safe’

Considered safe by experts and suitable for use in food

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7
Q

What is a recombinant protein?

A

A recombinant is a protein encoded by a gene that has been cloned in a system that supports expression of the gene

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8
Q

What is bacillus subtillis?

A

Gram positive and rod shaped bacteria

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9
Q

Why are there so few suitable expression vectors for b.subtilis compared to e.coli?

A

It is comparatively very poorly studied

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10
Q

When was the saccharomyces cerevisiae genome published?

A

1996

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11
Q

What does the Saccharomyces Genome Database contain?

A

All genomes and promoters annotated

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12
Q

What percentage of mammalian protein that were unable to be recombinant expressed in e.coli were expressed in sacchromyces cerevisiae?

A

70%

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13
Q

What does hyper-glycosylation in S.cerevisiae cause?

A

Reduced secretion rate and reduced drug efficacy

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14
Q

What is a methylotrophic yeast?

Give an example.

A

Can use methanol as its sole carbon source.

Pichia Pastoris

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15
Q

What and when was the first FDA protein to be produced by pichia pastoris?

A

Kalbitor (ecllantide)

In 2009

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16
Q

What expression host is commonly used to express HAS (most common protein in body)?

A

Pichia pastoris

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17
Q

Why is HAS required?

A

Used in surgery to prevent toxic shock

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18
Q

How many months is required for evaluate the production of a heterologous protein in mammalian cells?

A

3

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19
Q

What percentage of mammalian produced recombinant proteins are produced in CHO cells?

A

70%

20
Q

Name some proteins produced by Filamentous fungi.

A

Amylases, pectinases,lipases, cellulases, proteases, phytases, xylanases

21
Q

Name two filamentous fungi?

A

Aspergillus niger

Aspergillis oryzae

22
Q

Name a protein produced by Aspergilis Orzae.

A

Lactase (lactaid)

23
Q

What is the most common green algea used?

A

Chlamydomonas reinherdtii

24
Q

What was the first plant produced recombinant protein?

A 
Interferon (1986)
Serum Albium (1990)
25
Q

What plant was used to express Hepatitis B surface antigen in 1992?

A

Tobacco plant

Agrobacterium tumefaciens

26
Q

In 2011 human serum albumin (HSA) was produced in Oryza sativa up to 2.75 g Kg-1 of rice. What is a potential issue with this?

A

This rice is no longer edible. It is possible we are affecting other crops

27
Q

When producing enzymes what is the most important factor?

A

Cost
(also production speed)
Don’t need to worry about glycosylation

28
Q

What type of host is most popular enzyme production?

A

Microbial
Bacteria - smaller
Fungi/yeast - bigger

29
Q

What single protein was created as a vaccine for foot and mouth disease?

A

VP3

30
Q

What expression host was used to for expression of VP3?

A

E.coli

31
Q

What is VP3?

A

A virus-like particles

32
Q

When was VP3 first used for cattle and swine?

A

1981

33
Q

What and when was the first commercialised vaccine based on virus-like proteins licensed?

A

1986

a virus-like protein from Hepatitis B virus surface antigen (HBsAg)

34
Q

Transgenic plants are often used for VLPs. True or False?

A

True

35
Q

When was the first transgenic plant VLP produced? And what was it?

A

Hebatitis B virus

1992

36
Q

How does Cell Free Protein Synthesis work?

A

A cell is broken open and and cellular machinary is collectec and used to produce a protein invitro

37
Q

Is CFPS used on an industrial scale?

A

No

38
Q

Which lysates have been used for CFPS?

A 
E.coli
Wheat germ extract
Rabbit reticulocyte
Insect extract
CHO cells
HeLa cells
39
Q

What is the difference between saccharomyces cerevisiase and pichia pastoris glycosylation?

A

S.cerevisiase produced hyper-mannosylated proteins

40
Q

Why has pichia pastoris been fully humainsed?

A

To mimic human glycosylation pattern to s prevent rejection by host and improve drug efficacy

41
Q

Ye et al designed an optimal feeding strategy with humanised p.pastoris to produced a monoclonal antibody. What volume was this process scaled up to?
What was the yield

A

1200L

1.6gL^-1

42
Q

How can titre be positively increase in P.pastoris?

A

Increasing gene doasage

43
Q

Why isn’t increased dosage nit always a direct correlation to increase secretion?

A

Limitation of the secretory pathway (bottleneck)

44
Q

In p.pastoris how might burdens on the secretory pathway be lessened? Name 3 examples.

A 
Co-expression of folding chaperones
Specialised strains
Codon optimisation
Modified feeding strategies 
Alternative promoter
45
Q

What is a ScFv?

A

Single chain variable fragment

A fusion protein of the variable regions of an antibody connected with a short linker of between 10-25 amino acids

Biotechnology (13 decks)

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