Expression Hosts Flashcards
What percentage of biopharmacutical srae produced by Escherichia coli and Saccharomyces cerevisiae?
50%
What is a hybridoma?
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.)
What was the first recombinant protein produced?
Humulin
What expression host produced the first recombinant protein?
E.coli
What is an endotoxin?
Lipopolysaccharides -
large molecules consisting of a lipid and a polysaccharide
- found in the outer membrane of Gram-negative bacteria
What is a GRAS organism?
‘Generally Recognised As Safe’
Considered safe by experts and suitable for use in food
What is a recombinant protein?
A recombinant is a protein encoded by a gene that has been cloned in a system that supports expression of the gene
What is bacillus subtillis?
Gram positive and rod shaped bacteria
Why are there so few suitable expression vectors for b.subtilis compared to e.coli?
It is comparatively very poorly studied
When was the saccharomyces cerevisiae genome published?
1996
What does the Saccharomyces Genome Database contain?
All genomes and promoters annotated
What percentage of mammalian protein that were unable to be recombinant expressed in e.coli were expressed in sacchromyces cerevisiae?
70%
What does hyper-glycosylation in S.cerevisiae cause?
Reduced secretion rate and reduced drug efficacy
What is a methylotrophic yeast?
Give an example.
Can use methanol as its sole carbon source.
Pichia Pastoris
What and when was the first FDA protein to be produced by pichia pastoris?
Kalbitor (ecllantide)
In 2009
What expression host is commonly used to express HAS (most common protein in body)?
Pichia pastoris
Why is HAS required?
Used in surgery to prevent toxic shock
How many months is required for evaluate the production of a heterologous protein in mammalian cells?
3
What percentage of mammalian produced recombinant proteins are produced in CHO cells?
70%
Name some proteins produced by Filamentous fungi.
Amylases, pectinases,lipases, cellulases, proteases, phytases, xylanases
Name two filamentous fungi?
Aspergillus niger
Aspergillis oryzae
Name a protein produced by Aspergilis Orzae.
Lactase (lactaid)
What is the most common green algea used?
Chlamydomonas reinherdtii
What was the first plant produced recombinant protein?
Interferon (1986) Serum Albium (1990)
What plant was used to express Hepatitis B surface antigen in 1992?
Tobacco plant
Agrobacterium tumefaciens
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?
This rice is no longer edible. It is possible we are affecting other crops
When producing enzymes what is the most important factor?
Cost
(also production speed)
Don’t need to worry about glycosylation
What type of host is most popular enzyme production?
Microbial
Bacteria - smaller
Fungi/yeast - bigger
What single protein was created as a vaccine for foot and mouth disease?
VP3
What expression host was used to for expression of VP3?
E.coli
What is VP3?
A virus-like particles
When was VP3 first used for cattle and swine?
1981
What and when was the first commercialised vaccine based on virus-like proteins licensed?
1986
a virus-like protein from Hepatitis B virus surface antigen (HBsAg)
Transgenic plants are often used for VLPs. True or False?
True
When was the first transgenic plant VLP produced? And what was it?
Hebatitis B virus
1992
How does Cell Free Protein Synthesis work?
A cell is broken open and and cellular machinary is collectec and used to produce a protein invitro
Is CFPS used on an industrial scale?
No
Which lysates have been used for CFPS?
E.coli Wheat germ extract Rabbit reticulocyte Insect extract CHO cells HeLa cells
What is the difference between saccharomyces cerevisiase and pichia pastoris glycosylation?
S.cerevisiase produced hyper-mannosylated proteins
Why has pichia pastoris been fully humainsed?
To mimic human glycosylation pattern to s prevent rejection by host and improve drug efficacy
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
1200L
1.6gL^-1
How can titre be positively increase in P.pastoris?
Increasing gene doasage
Why isn’t increased dosage nit always a direct correlation to increase secretion?
Limitation of the secretory pathway (bottleneck)
In p.pastoris how might burdens on the secretory pathway be lessened? Name 3 examples.
Co-expression of folding chaperones Specialised strains Codon optimisation Modified feeding strategies Alternative promoter
What is a ScFv?
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
