Week 1 Flashcards
1
Q
What is a heterologous expression, give examples of heterologous hosts?
A
- Heterologous expression refers to the expression of a gene/part of a gene in a host organism that does not normally have this gene E.g. Bacteria (E. coli) E.g. Yeast E.g. Plants E.g. Baculovirus E.g. Cultured mammalian cells E.g. Animals
2
Q
What are some pros and cons of Bacterial Recombinant Protein Expression Systems?
A
Pros:
- Widely used
- Easily Manipulated
- Rapid to grow and cheap
- Many commercial vectors and tags to add to proteins to enhance purification
Cons:
- Proteins expressed are often insoluble (bacteria cannot produce large, complex and folded proteins meaning the recombinant protein may be defective)
- Bacteria cannot post-translationally modify proteins (bacteria cannot produce proteins reliant on phosphorylation, glycosylation etc.)
- There is a high endotoxin content that must be purified ou
3
Q
What is a fusion protein?
A
- Fusion proteins are proteins that have the DNA of the recombinant protein fused with the DNA of a purification tag
- This allows for the recombinant (fusion) protein to be extracted and purified from the heterologous host cell
- When the chimeric protein is expresed, the tag allows for the specific capture of the fusion protein (e.g. using an affinity column that binds the tag) and this fusion protein can then be eluted off
- The tag can then be, if neccessary removed using an enzyme
4
Q
How do glutathionine S-transferase tags work?
A
- The target protein cDNA is cloned into a vector (plasmid) downstream of a powerful promoter so lots of the protein will be synthesised
- The gene for the GST tag is also included downstream of the promoter
- Between these two genes a protease site is included
- The fusion proteins generated by the bacteria transfected with the plasmid are washed through a glutathione column where the GST tag of the fusion protein will bind
- These fused proteins are then eluted off
- An enzyme is added to cleave the protein from the GST tag
5
Q
What are some pros and cons of using tags in fusion proteins?
A
Pros:
- Can improve protein yield
- Can prevent proteolysis
- Facilitates protein refolding
- Increases solubility
- Increases ease of purification
Cons:
- Lower protein yields
- Alteration of biological activity
- Cleavage/removing the tag may require expensive proteases
6
Q
Where is recombinant protein expression targeted in bacteria?
A
- Cytosol: the cytosol is generally used for direct expression, however the cytosol is a reducing environment so proteins expressed cannot form S-S bonds
- Periplasm: the recombinant protein can be fused to proteins targeted for secretion so they are expressed in the periplasm which is a more oxidising environment. However the amount of protein expressed in the periplasm is very low and there is limited exporting ability for large proteins to be moved into the periplasm
7
Q
What is an inclusion body in E. coli?
A
- Inclusion bodies are dense particles that form within E. coli that contain precipitated proteins
- They occur because E. coli are unable to express and fold complex proteins such as mammalian proteins and these incorrectly folded proteins aggregate
Formation depends on protein synthesis rate (i.e. if it is being synthesised in very large quantities very quickly) and growth conditions - Protein refolding options exist but there are poor success rates
8
Q
How is Insulin synthesised recombinantly by Eli Lily?
A
- Insulin is expressed recombinantly in E. coli bacteria
- The issue with E. colli is that there is no secretory pathway for processing and no S-S capacity in the bacteria cytosol
- Researchers instead express the A and B chains seperately and then purify them and then bring them together in an oxidising solution where they fold into their native confirmation
9
Q
What are the pros and cons of yeast recombinant expression systems?
A
Pros:
- They have a secretory pathway
- Cheap to culture
- Eukaryotes
- Can get S-S bond formation
- Simple to genetically manipulate
Cons:
- Not good for mammalian glycoproteins as gylcosylation patterns differ in yeast
10
Q
Why must EPO be synthesised in mammalian cell cultures?
A
- EPO is a hormone synthesised by the kidney that stimulated RBC synthesis
- Glycosylation of EPO is required for EPO action
- Around 50% of all eukaryotic proteins are glycosylated (usually done in the ER)
- Mammalian cell culture used so the correct glycosylation can take place
11
Q
How does Mammalian tissue culture work?
A
- Uses a growth media, extra factors e.g. FCS and primary and transformed mammalian cell lines
- The cells used are usually CHO (Chinese hamster ovary) cells
- Capable of phosphorylation, glycosylation and very reliable protein folding
12
Q
What are the mechanisms of action of antibodies?
A
- Ligand blockade:
- Antibody binds to ligand and takes it out of action so it can no longer bind the receptor - Receptor blockade:
- Antibody binds to receptor and blocks it from binding to the ligand - Receptor down regulation:
- The antibody binds to the receptor and causes it to be endocytosed into the cell - Depletion:
- The antibody binds to the surface of the signalling cells and activates immune responses e.g. complement, phagocytes, to kill the signalling cell - Signalling Induction:
- The antibody can bind receptors dimerising them and activating them in the absence of the ligand
13
Q
What is a polyclonal antibody, how are they obtained?
A
- Polyclonal antibodies: a collection of antibodies from different B cells that recognise different antibodies
- They are gotten from an animal that has been immunised against a certain antigen and the antibodies are collected from the animal’s sera
14
Q
- What is a monoclonal antibody?
- How are they produced?
A
- An animal e.g. mouse, is immunised against a particular antigen and spleen cells that contain antibody producing B cells are isolated from the animal
- These primary spleen cells have a limited life span so they are fused with an immortal cell (myeloma) producing an immortal antibody producing cell (hybridoma)
- The hybridomas must be selected from over left over myeloma cells:
- Myeloma cells lack an enzyme needed for the nucleotide salvage pathway
- Standard de novo synthesis of nucleotides is inhibited so cells must use the salvage pathways to survive
- As the myelomas cannot undergo this nucleotide salvage pathway they die and only the hybridomas survive
- The hybridomas are then seperated into single clones in seperate tubes and they will produce monoclonal antibodies which can then be assessed for recognition of the antigen
15
Q
List the types of monoclonal antibodies from most to least likely to elicit a human immune response:
A
- Mouse
- Chimeric: has mouse variable regions
- Humanised: has mouse CDR regions
- Human: genetically human
16
Q
How are chimeric monoclonal antibodies made?
What is an example?
A
- Mouse variable light chains and human constant light chains are expressed into the same plasmid
- Mouse variable heavy chains and human constant heavy chains are expressed into the same plasmid
- These two plasmids are then cloned into a myeloma cell
- The myeloma cell will then secrete the chimeric antibody
e. g. Rituximab:
- A chimeric anti-human CD20 monoclonal antibody used to treat non-Hodgkin’s lymphoma
