Expression Hosts

Question 1

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

Answer 1

50%

Question 2

What is a hybridoma?

Answer 2

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.)

Question 3

What was the first recombinant protein produced?

Answer 3

Humulin

Question 4

What expression host produced the first recombinant protein?

Answer 4

E.coli

Question 5

What is an endotoxin?

Answer 5

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

Question 6

What is a GRAS organism?

Answer 6

‘Generally Recognised As Safe’

Considered safe by experts and suitable for use in food

Question 7

What is a recombinant protein?

Answer 7

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

Question 8

What is bacillus subtillis?

Answer 8

Gram positive and rod shaped bacteria

Question 9

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

Answer 9

It is comparatively very poorly studied

Question 10

When was the saccharomyces cerevisiae genome published?

Answer 10

1996

Question 11

What does the Saccharomyces Genome Database contain?

Answer 11

All genomes and promoters annotated

Question 12

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

Answer 12

70%

Question 13

What does hyper-glycosylation in S.cerevisiae cause?

Answer 13

Reduced secretion rate and reduced drug efficacy

Question 14

What is a methylotrophic yeast?

Give an example.

Answer 14

Can use methanol as its sole carbon source.

Pichia Pastoris

Question 15

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

Answer 15

Kalbitor (ecllantide)

In 2009

Question 16

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

Answer 16

Pichia pastoris

Question 17

Why is HAS required?

Answer 17

Used in surgery to prevent toxic shock

Question 18

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

Answer 18

3

Question 19

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

Answer 19

70%

Question 20

Name some proteins produced by Filamentous fungi.

Answer 20

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

Question 21

Name two filamentous fungi?

Answer 21

Aspergillus niger

Aspergillis oryzae

Question 22

Name a protein produced by Aspergilis Orzae.

Answer 22

Lactase (lactaid)

Question 23

What is the most common green algea used?

Answer 23

Chlamydomonas reinherdtii

Question 24

What was the first plant produced recombinant protein?

Answer 24

Interferon (1986)
Serum Albium (1990)

Question 25

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

Answer 25

Tobacco plant | Agrobacterium tumefaciens

Question 26

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?

Answer 26

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

Question 27

When producing enzymes what is the most important factor?

Answer 27

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

Question 28

What type of host is most popular enzyme production?

Answer 28

Microbial Bacteria - smaller Fungi/yeast - bigger

Question 29

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

Answer 29

VP3

Question 30

What expression host was used to for expression of VP3?

Answer 30

E.coli

Question 31

What is VP3?

Answer 31

A virus-like particles

Question 32

When was VP3 first used for cattle and swine?

Answer 32

1981

Question 33

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

Answer 33

1986 | a virus-like protein from Hepatitis B virus surface antigen (HBsAg)

Question 34

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

Answer 34

True

Question 35

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

Answer 35

Hebatitis B virus | 1992

Question 36

How does Cell Free Protein Synthesis work?

Answer 36

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

Question 37

Is CFPS used on an industrial scale?

Answer 37

No

Question 38

Which lysates have been used for CFPS?

Answer 38

``` E.coli Wheat germ extract Rabbit reticulocyte Insect extract CHO cells HeLa cells ```

Question 39

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

Answer 39

S.cerevisiase produced hyper-mannosylated proteins

Question 40

Why has pichia pastoris been fully humainsed?

Answer 40

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

Question 41

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

Answer 41

1200L | 1.6gL^-1

Question 42

How can titre be positively increase in P.pastoris?

Answer 42

Increasing gene doasage

Question 43

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

Answer 43

Limitation of the secretory pathway (bottleneck)

Question 44

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

Answer 44

``` Co-expression of folding chaperones Specialised strains Codon optimisation Modified feeding strategies Alternative promoter ```

Question 45

What is a ScFv?

Answer 45

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