What is the purpose of biological expression systems?
To produce proteins.

Why express heterlogous proteins?
Proteins are difficult to purify and obtain in large quantities. Expressing proteins fulfills both needs. It is also less expensive once an expression system has been set up.

What are expression systems?
Expression systems are vectors and host cells which are used to produce a protein. An expression system may be cell-based or cell free.

What the necessary step to produce a protein?
1. deciding which polynucleotide sequence to introduce
2. introducing mRNA or DNA into a cell-free extract
3. introducing DNA into a cell
4. extracting and purifying the desired proteins

How can we produce a novel protein?
It is possible to mutate tRNA genes of any organism i.e. modify the genes which code for tRNA. The goal of this exercise is to stimulate or suppress an activity. This technique allows the addition extra amino acids into the system and may result in the production of novel proteins. Protein splicing can take place after translation.

What are the advantages and disadvantages of cell-free systems?
Advantages
• bypassing cellular metabolism means that label incorporation can be more efficient
• highly toxic gene products can be made in this way
Disadvantages
• Although not present, cellular chaperones can be added to the system
• PTM that require cellular structures cannot be made

What are the advantages and disadvantages of cell-based systems?
Advantages
• can be adapted for producing very large quantities of protein at relatively low cost
• cell can be used to make isotopically labeled proteins
• can incorporate PTM
• can use cell-based expression to look at the function of a protein in the context of a living cell
• expression can be made to last as long as the cell culture lives.
Disadvantages
• Time consuming: cloning of target DNA, introduction of vector, growth of cell culture, and induction
• Proteins need to be extracted and purified from the cells

What is transformation?
Genetic alteration of a cell after uptake of foreign DNA.

What is transfection?
Transformation of animal cells.

What is transduction?
Transduction is the process by which bacterial DNA is moved from one bacterium to another by a virus.

How do you choose an expression system?
The choice depends largely on your needs. You cannot, for example, produce antibodies in a bacteria.

What is a vector?
Traditionally in medicine, a vector is an organism that does not cause disease itself but which spreads infection by conveying pathogens from one host to another. Mosquitoes do not cause malaria but spread the pathogen. A virus itself may serve as a vector, if it has been re-engineered and is used to deliver a gene to its target cell. A "vector" in this sense is a vehicle for delivering genetic material such as DNA to a cell by a process called transduction. In genetic engineering vectors are usually plasmids or phages.

What is a plasmid?
Plasmids are autonomously replicating extra chromosomal DNA molecules. Plasmids often contain genes that confer a selective advantage to the bacterium harboring them, e.g. the ability to make them antibiotic resistant. Every plasmid contains at least one DNA sequence that serves as a starting point for DNA replication, enabling the plasmid DNA to replicate itself independent of the chromosome.

Plasmids used in genetic engineering are called vectors. They are used to transfer genes from one organism to another and typically contain a genetic marker conferring a phenotype that can be selected for or against. Most also contain a polylinker or MCS (multiple cloning site), which is a short region containing several commonly used restriction sites allowing easy insertion of DNA fragments at this location.

What are lentiviral vectors?
Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell. HIV is a very effective lentiviral vector because it has evolved to infect and express its genes in human helper T cells and other macrophages. The only cells lentiviruses cannot gain access to are quiescent cells (in the G0 state) because this blocks the reverse transcription step. Reverse transcription is the process of coverting RNA to DNA.

What is a flag-tag?
FLAG-tag is a polypeptide protein tag that is added to a recombinant expressed protein. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits.

What are inducible and constitutive promoters?
A promoter is a DNA sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. Inducible promoters can be artificially activated or inhibited. Expression of constitutive promoters is not controlled by endogenous factors. Constitutive promoters are used for repressor proteins.

Describe lentiviral infection
1. The virus attaches itself to CD4 and co-receptor of the T helper cell membrane.
2. It injects the viral core into the cytoplasm.
3. Virus loses its membrane, consisting of nucleoproteic complex.
4. This complex enters the nucleus, does reverse transcription and inserts itself into the genome.

How can we insert a vector into a cell?
- Precipitation at the cell surface
- electroporation
- lipid vesicules
- using projectiles
- using viral vectors

How do we detect or purify a protein?
- Separate the lysate using electrophoresis
- Western blot

Amino acids
RW - H donors
NQST - H donors and acceptors
KDEYH - donor or acceptor based on their ionization state

Which amino acids are involved in electrostatic interactions? Hydrophobic
Electrostatic: polar - SCN WYTH
Hydrophobic : non polar - FW MAIL PGV

What is covalent capture
We place a Cys or Thr at the end of a polypeptide chain in solid phase with aldehydes. This helps purification as the lateral chain would not grow.

Ribosome Display
http://www.bioc.uzh.ch/plueckthun/slide_shows/Slides/ribo/ppframe.htm

How to cut & paste transgenes?
Restriction enzymes

How are codons read?
The adaptor in protein synthesis is tRNA. tRNA molecules contain an amino acid attachment site and a template recognition site (anticodon). Amino acid chains are assembled by ribosomes according to instructions originally on the DNA template copied on to mRNA transcript.

What are the start and stop codons?
start: AUG
stop: UAA, UAG, UGA

What are suppressor mutations?

List cell-based systems
Prokaryotic Transformation - concern bacteria, precipitation of DNA at the surface, electroporation
Prokaryotic Transduction - uses viral vectors
Eukaryotic Transduction - uses viral vectors
Eukaryotic Transfection - DNA at surface, electroporation, precipitation, projectiles, etc.

What is a polylinker or MCS?
A polylinker or MCS (multiple cloning site) is a short region containing several commonly used restriction sites allowing easy insertion of DNA fragments at this location.

What is Ori?
The origin of replication (also called the replication origin) is a particular DNA sequence at which DNA replication is initiated. DNA replication may proceed from this point bidirectionally or unidirectionally.

What are the specific features of an expression vector?
- Promoter - inducible such as lac operon
- Origin of Replication
- Transgene
- Tags to help purification
- IRES - to allow expression of multiple genes
- MCS - for restriction enzymes
- antibiotics resistance genes

What is an ORF?
The section of genome between start and stop sequences. The section which could potentially code for something.

What are the steps in Western Blot?

Draw a vector

What can we add to the vector? Why?
IRES: to express multiple genes
neoR:
tag or flag: to facilitate purification

When a protein is cloned, how do we verify that the protein is expressed?
WB and MS

How do we purify?
2D, chromatography

Explain affinity chromatography
1. An antigen is added to an immunoabsorbent.
2. Proteins are passed over the immunoabsortbent. Desired antibodies will non-covalently bond with antigen. Everything else will flow through.
3. Use salt of urea for elution to separate antigens and antibodies.
4. Dialysis to remove the elution materials.

Ion exchange chromatography

Reverse Phase chromatography

Gel-filtration, size-exclusion chromatography

Why are antibodies important?
Protein detection: FACS, WB, ELISA
Protein purification: affinity chromatography
Protein localization: immunofluorescence

FACS
Fluorescent antibodies can also be used with a fluorescence-activated cell sorter (FACS) to identify and even physically separate different cell populations according to the antigens expressed on their surfaces. A FACS is a flow cytometer designed to detect fluorescent light stimulated by the laser beam. Cells binding fluorescently tagged monoclonal antibodies directed against specific cell surface CD antigens, for example, will emit fluorescent light as they pass through the cytometer's laser beam. The relative intensity of emitted fluorescent light correlates with the amount of antibody bound to the cell surface which in turn correlates with the level of CD marker expression by the cell. With proper combinations of antibodies with different fluorescent tags, specific cell populations can be sorted and even isolated from a complex mixture of cells.

ELISA
Enzyme-Linked Immunosorbent Assay (ELISA) is used to detect presence of an antibody of antigen. It uses two antibodies coupled to an enzyme, one of which is specific to the antigen. It the first antibody attaches to an antigen, the second would produce a detectable chromogenic or fluorogenic signal.

immunofluorescence
1. Add your primary antibodies which will bind to a certain protein.
2. Add your secondary fluorescent antibodies which will bind to primary antibodies.
3. View with a microscope.

Monoclonal antibodies
Monoclonal antibodies are all identical, produced by clones of a single antibody-producing cell. They recognize one specific epitope. Producing mAb requires immunizing an animal, usually a mouse; obtaining immune cells from its spleen; and fusing the cells with a cancer cell (such as cells from a myeloma) to make them immortal, which means that they will grow and divide indefinitely. A tumor of the fused cells is called a hybridoma, and these cells secrete mAb. The development of the immortal hybridoma requires the use of animals; no commonly accepted non-animal alternatives are available. An investigator who wishes to study a particular protein or other molecule selects a hybridoma cell line that secretes mAb that reacts strongly with that protein or molecule. The cells must grow and multiply to form a clone that will produce the desired mAb.

Western Blot
A sample is subjected to electrophoresis on an SDS-PAGE. The resolved proteins are transferred onto the gel. An antibody specific for an antigen is added. A second antibody which is specific to the first antibody is added and rinsed. If a label is detected, we have the antigen, otherwise we don’t.

Polyclonal antibodies
Most antigens have several epitopes. Polyclonal antibodies are heterogeneous mixtures of antibodies, each specific for one of the various epitopes on an antigen.
1. Immunize animal
2. Purify IgG from the serum

Antibody Structure
Antibody Fragments
CDR - complementarity determining regions

What is ribosome display?
Ribosome display is a technology for the in vitro selection and evolution of the very large protein libraries. The entire procedure is performed in vitro, without using cells at any step. Steps:
1. Isolation mRNA
2. Reverse transcribe mRNA to DNA
3. Amplify with PCR
4. in vitro transcription to produce mRNA
5. in vitro translation. Since there is no stop codon, native proteins are tethered to the ribosome
6. selection on surface bound target
7 dissociation of mRNA-ribosome protein complexes. Repeat from step 1.

Features of an expression vector?
Packaging plasmid
Envelope plasmid
How to get nucleic acid into the cell
Flow diagram for getting a transgene into a cell
How do you get the transgene in?
How do you switch it on?
Pros and cons of different systems
Checking expression and adjusting parameters
Purification of the expressed protein
1. Protein Expression:
- Isolation and characterization of recombinant proteins
3. Protein/peptide chemical synthesis
4. Protein arrays
5. Protein Interactome
- Methodology
- Protein-protein interactions
- Protein-polynucleotide interactions
- Interaction with other biomolecules
6. Interactome bioinformatics:
- Definition of interactome (systems biology)
- Practical approaches
- Prediction: description of different algorithms
- Modelling interactions, pathways, cellular systems
- Challenges for bioinformaticians (prediction and interpretation of complex data)
- Visualization tools
7. Interactome databases:
- Interaction databases
- Pathway databases
- Information content and overlap
- Limitations

Antibodies
Monoclonal and polyclonal antibodies: creation, how to create antibodies (lympho-B)
How to create antibodies without animals (phage and ribosome display)
Ribosome display: we start with the database of 2 billion sequences of antibodies. Quand on a notre complexe mRNA-ribosome-proteine on fait un patenting et non un screening pour trouver le bon Ab dirige contre notre Ag, et ensuiter on recupere le mRNA qui correspond a cette proteine et on y induit des mutations pour augmenter l’affinite.
Antibodies : structure, production
Humoral response, cellular response, CDR
Antibody-antigen steric complementarity
Bonds: Hydrophobic interactions, Electrostatic interactions,
Which amino acids with related to which bonds
Difference between monoclonal and polyclonal antibodies - molecular aspects of this question: gene recombination, variable parts, CDR, maturation affinity.
Primary and secondary immune response
How many cDNA and antibodies are in a bank of antibodies
Structure of antibodies and antigens. Draw. Interactions. Amino acids involved. (steric complementarity, RW donor, NQST donor-acceptor, DEKYH donor-acceptor, electrostatic interactions (DE and KRH), hydrophobic interactions (FWYMAIL).
Why entropy in when two hydrophobic regions come closer. Because normally the polar molecules and water are arranged on the surface for the hydrophobic region; when they leave, the entropy increases in that region.

Peptide Synthesis
Non-natural amino acids (stop suppression, solid phase chemistry)
How to create a protein which contains sys tRNA muted (long et cher) or chemical synthesis (long and expensive but limited in size)
Peptide synthesis: reaction, rendement, purification, etc.
Properties of amino acids
Different types of chromatographies

Network model: random, scale free, hierarchical, p(k) and c(k)
Origins of scale free in nature
IG, EPR and PVM. Which one is most used with networks (EPR)? Why?
Computation method for PPI? How it works
Draw a graph of K vs. P(k) for scale free. Duplication hypothesis and hubs.

Experimental methods
Coimmunoprecipitation
Scale free networks: details, comparison, robustness (tolerance to error and resistance to attacks) in comparison with random networks
Y2H: false positives, pros and cons
A method to detect antibodies (CoIP) with advantages and disadvantages

Databases
Format PSI-XML
Some entries come from experimentation, others from prediction

PPI prediction
A method for PPI and explain
Similarity between phylogenetic trees. MSA of proteins and distance matrix.
Domain interactions method for ppi IntAct
Computational methods for PPI