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
17
Q
How are antibodies generated using Phage display libaries?
A
- B cells from a population can be taken and then the antigen recognising portions of the heavy and light chains can be amplified using PCR
- These antigen recognising molecules are bound together at random and may recognise antigen (as scFV)
- The bound scFv are then cloned into bacteriophages fused to coat protein DNA
- The bacteriophage will then infect bacteria and the scFv gene along with the other genes will be translated and then these fusion proteins (a cot protein + the scFv) will be sent to the periplasm and then displayed on the surface of the phage that is produced by the infected cell
- The target receptor or cell is then exposed to the phage library; the phage that binds will be the phage expressing the specific antibody or fragment
- The specific phage can then be transfected back into E. coli to replicate and become more selective
- The DNA of the Fc fragment of Fab can then be screened for specificity
- The DNA can then be put into a human antibody scaffold and be placed into a myeloma cell for mass production
18
Q
How can guided selection be used to generate fully human antibodies against TNF?
Give an example of a human mAb
A
- A mouse monoclonal antibody against TNF can be used to guide the selection of a human antibody against TNF
1. The heavy chain of the mouse anti-TNF antibody is paired with a human light chain repertoire to make a phage library
2. In parallel the mouse light chain anti-TNF antibody is paired with a human heavy chain repertoire in another phage library
3. Both libraries are selected on antigen (human TNF)
4. The resulting selected human and heavy and light chains are then paired together and tested.
e.g. Adalimumab/Humira is the first human Mab against TNF
19
Q
What is personalised medicine?
A
Personalised medicine has been defined as: “a medical model that proposes the customization of healthcare — with medical decisions, practices, and/or products being tailored to the individual patient based on their predicted response or risk of disease”
20
Q
What is genomic medicine?
A
- A medical discipline that involves using genomic information about an individual as part of their clinical care
21
Q
What is Rubraca?
A
- Rubraca is a poly ADP-ribose polymerase (PARP) inhibitor that aims to reduce the mitosis of tumour cells
- It is used to treat women who have advanced ovarian cancer and who’s tumours have a specific BRCA mutation
22
Q
What is Epclusa?
A
- A combination of sofosbuvir and velaptasivir used to treat hepatitis C
- It treats most genotypes of hepatitis C
23
Q
What is tecentriq?
A
- A humanised monoclonal antibody that binds to PD-L1 and blocks the interaction between PD-L1 and PD-1 and B7.1 receptors
- It is this interaction between PDL1 on cancer cells (a checkpoint protein that normally restrains the immune response) and PD-1 on T cells that prevents T cells from killing the tumour cells
- Tecentriq binds and inactivates PD-L1 to allow for the T cell killing of tumour cells
- Used for metastatic non-small cell lung cancer
- Patients with higher PD-L1 expression in their tumour cells showed a much greater response
24
Q
What is exondys 51?
A
- An anti-sense oligonucleotide used for the treatment of DMD in patients with a confirmed mutation of the DMD gene that is amenable to exon 51 skipping
- The anti-sense oligonucleotide binds to the intron-exon boundary on pre-mRNA and prevents the spliceosome from binding meaning the exon (51 in this case) will be skipped and not included in the mRNA transcript
- Skipping exon 51 can help bring the transcript of dystrophin back into reading frame so a more functional protein can be synthesised
- Not shown to have clinically significant effects
25
What is gene therapy?
- The aim of gene therapy is to correct a mutation in a gene by introducting a wildtype copy of a gene into cell of interest e.g. by a viral vector such as adenovirus or another retrovirus
26
What is genome editing?
| What are the 3 main strategies?
- Genome editing involves targeting specific cells and correcting the genome itself rather than introducing a wildtype gene via a vector
- Generally involves modifying genes following the introduction of double stranded breaks by:
1. ZFNs
2. TALENs
3. CRISPR/Cas9
27
What is non-homologous end joining?
- This strategy involves causing a double stranded break in a gene and then letting the cell's own DNA repair mechanisms correct the gene
- No donor DNA is provided so the cell's DNA repair mechanisms will either add or remove nucleotides forming indels (small insertions or deletions of 5-10 nucleotides)
- In this way NHEJ knocksout a gene
28
What is homologous end joining?
- After a double stranded break is introduced donor template DNA is introduced into the cell
- The donor template DNA has homologous arms to allow for its introduction via crossing over into the broken DNA
- The donor DNA can have variable regions between the homologous end- in this way a corrected sequence of the DNA can be introduced into cells
29
What is a zinc finger nuclease?
- ZFNs work by having a series of proteins called zinc fingers that recognise nucleotides (each zinc fingr protein recognises a set of 3 nucleotides)
- Creating a fusion protein with multiple zinc fingers can target a specific sequence of DNA adjacent to the site where the ds break is intended
- Fused to the zinc finger proteins is Fok1 nuclease
- When the zinc finger domains bind both strands of the DNA the Fok1 nuclease proteins are brought together so they can dimerise and become activated and then cleave both strands of DNA
- NHEJ or HR can then take place
30
What is a transcription activator-like effector nuclease?
- TALENs are a series of protein modules that bind individual nucleotides
- Using cloning TALENs are joined together as fusion proteins that recognise a specific sequence of DNA
- Fused to this series of TALENs is Fok1 nuclease
- When both the left and right TALE recognise their specific sequences and bind the Fok1 nucleases are able to dimerise and become activated and cleave the DNA
- NHEJ and HR can then take place
31
How does CRISPR/Cas9 work?
- A target sequence that is complementary to the guide RNA must have a PAM (protospacer adjacent motif- NGG) just downstream of it
- The first RNA section of the gRNA anneals to the target sequence via nucleotide-nucleotide binding and the other section of the gRNA is attached to Cas9 nuclease
- The Cas9 nuclease then cleaves the DNA at the nuclease domain (a few bases upstream of the PAM)
32
What are some advantages of CRISPRs over ZFN/TALENs?
- CRISPR binds to target DNA via classic base pairing rules
| - Making custom gRNA is much easier, cheaper and quicker than generating ZFs or TALEs
33
What are some disadvantages of CRISPRs compared to ZFNs/TALENs?
- The overall recognition sequence of CRISPs is small making off target effects a potential issue (TALENs have much lower off-target effects)
34
How is genome editing used?
1. Basic Research: generating animal models and cell lines
2. Screens to identify new therapeutic targets
3. Therapeutics: ex vivo and in vivo
35
How is CRISPR/Cas9 used to generate cell lines for research?
- This process uses a plasmid called Puro which can be put into a mammalian cell
- The plasmid contains a U6 promoter, then a cloning site where the DNA encoding the specific annealing gRNA sequence and the scaffold portion of gRNA are inserted
- Downstream of the U6 promoter and gRNA sequences is DNA encoding a modified Cas9 nuclease
- The plasmid also contains DNA for a GFP marker
- The cells can be sorted using the GFP marker
- Transfected cells can then be screened the the targeted gene can be sequenced to determine if it has been altered e.g. looking for indels if NHEJ is taking place
- In this way gene editing can be done to cell lines
36
How is CRISPR/Cas9 used to generate animal models for research?
- A specific gRNA and Cas9 sequence is inserted into a zygote in order to target a gene of interest through NHEJ (to generate a knockout) or HR (to generate a transgenic organism)
- The edited zygotes can be inserted into a surrogate
- This is a cost effective and quick means of generating knockout and transgenic mice
- If HR is used a small tag/GFP can be added to the donor DNA so transgenic organisms can be easily identified
37
How is CRISPR/Cas9 used to screen for new therapeutic Targets?
E.g. West-Nile-Virus causes massive neuronal death but it was not understood how the virus activated downstream death pathways in neurons:
1. A library of gRNAs targeting all genes of the human genome was generated
2. The gRNA was cloned into and expressed in lentiviruses
3. Human cells were then infected with a lentivirus (containing one gRNA that targets one gene)
4. A plasmid containing Cas9 was then added to these cells
5. Therefore a single gene knockout cell for each gene was created
6. There single gene knockout cells were then challenged with West-Nile-Virus
7. Surviving cells were grown up and amplified via PCR and sequenced, this revealed the genes that were required by the West-Nile virus to initate cell death
38
How does ex vivo somatic therapeutic gene editing work?
- Involves the removal of the cells from the body
- The genes of these cells are then modified
- The cells are then transplanted back into the patient to treat disease
- Best done with blood cells such as T cells
Limitations:
- Potential for mutations of cells in cell culture
- The edited cells must be needed in low levels to overcome the disease phenotype and have a fitness advantage over the unedited cells
39
How does in vivo somatic therapeutic gene editing work?
- Involves either systemic or targeted approaches
e. g. Systemic approaches involve injecting gRNA and Cas9 directly into muscle tissue to target muscle cells
e. g. Targeted approaches involve injecting gRNA and Cas9 directly into the portal vien to target the liver, or putting gRNA and Cas9 into a virus that infects a particular tissue
Limitations:
- Difficult to target the right organ
- The lifespan of the cells targeted
- Homologous recombination does not occur in non-dividing cells
- The edited cells must be needed in low levels to overcome the disease phenotype and have a fitness advantage over the unedited cells
40
Describe how ZFNs were used to treat HIV:
- CCR5 is a T cell co-receptor for HIV-1 virus,
- Individuals with a loss of 32 aa in CCR5 gene are resistant to HIV-1 infection
- The ex vivo strategy was to take patient T cells and edit them with ZFNs and NHEJ to induce a 32 aa deletion mutation
- The edited T cells were reintroduced and reduced viral load
41
How were TALENs used to treat B-cell leukemia?
- The patient received T cell therapy which used modified T cells to target the cancerous cells
- T cell therapy usually requires autologous cells however B cell lymphoma cells cannot have their T cells harvested so T cells from different people were taken and modified using TALENs so they would not be recognised by the patients immune system
