Bernadette Byrne Flashcards
Main overarching idea behind Byrne Lecture series?
Learning about how to produce/express large quantities of a desired protein so that it can undergo further downstream analysis
What are the main elements in an expression vector (plasmid)?
Genetic elements involved in expression of plasmid-encoded protein…
Basically, protein gene inserted into a plasmid vector which is then introduced into a host which can subsequently produce the protein.
Outline the function of the different elements in a expression vector?
- Promoter –> control production based on kinetic information from the rest of the vector sequence (rate of production) + it’s also the region where RNA poly binds
Different promoters with different characteristics i.e. inducible – we can switch on/off to obtain adequate amount of protein - binding of RNA poly to promoter that drives production
- Operator –> regulatory/control region - ensures that the protein is produced at an optimal time during the cells life - proteins bind to the operator in order to inhibit/enable RNA poly promoter binding - controls production of protein.
- RBS –> sequence for ribosomal binding site marks the start of RNA transcript –> where ribosome binds
- ATG - Start codon to indicate start site of gene
- Gene of interest - what we inserted in order to produce
- STOP codon - translation should stop
- Terminator - Marks the end of the RNA transcript transcription stops
Why are E. Coli promoters useful?
They are inducible - meaning they can be switch on/off
Apart from our protein of interest what else is typically added to the expression vector?
Selection marker - indicates that the plasmid has integrated
For example in E. Coli
Antibiotic resistance markers are used to select colonies that produce protein of interest
What is an example of a useful E. Coli Inducible promoter?
What are the different phases of E. Coli culture growth?
Lag phase –> cell are dividing at a slow rate
Exponential phase –> rapid growth
Stationary Phase –> no net growth
Decline/death phase –> nutrients used up
At what point on our E. Coli growth is it best to induce production of our protein?
Optimal - target cells when there are growing the quickest/abundant of nutrients –> exponential phase roughly a OD of 0.6
Furthermore, once we introduce a inducer we lower the temperature - prolong the exponential phase
What might compete with expression of target protein?
Proteins involved in growth, cell cycle regulation, housekeeping proteins, etc. (cells own cellular processes) –> proteins compete for resources in order to be expressed.
Solution?
We can reduce temperature during the exp. Phase reduce growth rate –> divert resources to POI expression.
What does autoinduction refer to?
Autoinduction –> relies on consumption of carbon source in media - once source has been used up the repressor has been removed (assuming that the carbon source is a repressor in the first place) –> allowing for induction
What is one of the main problems with a normal E. Coli expression system? How can this problem be overcome?
What is one of the main advanatages of using an E. Coli expression system?
Advantage of using E. Coli –> cheap and easy to use
The attached table shows different E. Coli expression systems used in the table, outline the benefit of each strain.
Other ways to optimise expression of protein of interest?
Before we can start cloning our gene of interest into a plasmid, what do we have to do?
We need to amplify our gene of interest –> PCR is used to amplify the quantity of DNA we have.
The following table shows different possible DNA polymerases that are used in PCR - answer the following questions…
What are the benefits of taq polymerase? Why is it not used as frequently?
What does processivity refer to?
What is the best option to use?
What is one of the main benefits of using taq polymerase in PCR? But what is one downfall which you need to watch out for?
Benefit –> taq leaves a 3’ A overhang which can be useful for gene insertion into a plasmid
Problem?
Insertion in the plasmid is not directional –> so you must sequence the plasmid to check for colonies with the correct orientation.
What is TA-cloning with topoisomerase? Why is it useful?
TOPO-TA
- Plasmid - overhang is created with topoisomerase I, an enzyme that recognizes 5′ (C/T)CCTT 3′ - does not release as it remains bound to the 3’ end
- Insert - Use Taq polymerase to create a A overhang
Mix
Result - nucleophilic attack by insert on plasmid overhang allow for ligation to occur
How is traditional cloning perfomered (intertion of PCR product into plasmid)?
Limitations of traditional cloning?
What is an alternative to traditional cloning which does not require ligation?
Outline a possible procedure that is used by ClonTech for Ligation-independent Cloning of PCR fragments?
What is Gateway technology - Holding vector? Why is it useful?
What is a fusion protein?
This is when we express a protein linked to a tag
We may want to add a tag used for detection, localization, purification
NOTE
Must include a protease recognition site so that we can remove the tag once the protein has been obtained so that our protein resembles the native protein as much as possible
What are some examples of commonly used tags? What are they used for?
Is the protein present? –> can also be figured out by using a western blot - uses antibodies for particular protein
What are some examples of protease enzymes used to cleave off fusion tags?
What is an example of an E. coli expression vector that is useful?
T7 promoter
RBS
ATG
His-tag
Xpress Epitote
Enterokinase Cleavage site
Multiple Cloning site
T7 terminator
What is one thing we need to remember when inserting our gene of interest into a vector?
When we run PCR with our gene of interest we use primers that contain our restriction site, so upon addition of our restriction enzyme we obtain our fragments with a specific known overhang.
Our vector which we use to insert our GOI contains multiple restriction sites from which we can choose from but…
Wee need to know the whole sequence of the vector to ensure that when we do insert of GOI at a specific site it will be in-phase with all the regulatory regions.
How can we minimze any additional bases in our transcribed transcript from our cloning vector?
It is important to note that any sequence that is present after the ATG initiation site and before the point of insertion in the MCS will be present on the N-Terminus of the protein
Thus, when deciding where to insert we want to minimize the amount of extra bases/A.A located at the N-terminus
Example below, inserting into BamHI site would be the most optimal.
How do we design our forward and reverse primers when amplifying our GOI using PCR?
These are example gels from the His-Tagged cyanoP protein purification using Ni column –> Outline what each column represents in each gel.
Why are bacterial expressions systems the most commonly used?
Expression systems –> Bacterial systems are normally the most commonly used expression system because they are easy to use, cheap, large range of tools, expression is quick
What are the reason why a bacterial expression system may not be useful/suitable?
Apart from bacterial systems, what else can be used?
These are the most common:
- Yeast - Pichia or Saccharomyces cerevisiae
- Insect - Viral based infection
- Mammalian - Transient or stable
- Cell free
Things to consider when choosing a system?
- Source of the gene - Would it be useful to express mammalian proteins in E. Coli? Not really useful to have a eukaryotic protein expression system
- Specific protein requirements - molecules, cofactors, chaperones, etc.
- What is the downstream application? - what are you going to do with the protein –> structural studies? Examining behavior of protein in cell? Etc.
General speaking, how is a Pichia Yeast expression system used?
Characterisitics of a Pichia vector?
One benefit and one problem of using the Pichia expression system?
Benefit - Pichia can grow to high cell density
In E. Coli the O.D. normally reaches 0.6 before we induce However, in Pichia it is much higher - into the hundreds
Problems with Pichia system?
The number of multiple cloning site is comparatively limited - not a large number of tools to work with.
How does the process of incorporating the plasmid into the Pichia genome work?
Note – this is not plasmid-based expression as we integrate the plasmid into the host genome + Can’t control how many copies insert
Most likely have multicopy insertion - this can be an advantage but more integrated DNA doesn’t mean more production
What setup is used when growing and expressing in Pichia systems?
What are some variables that are analysed in a Bioreactor?
What is one main issue associated with working with yeast cells? How is it overcome?
Is it possible to use Saccharomyces cerevisiae as an expression system instead of Pichia, are there any benefits?
Outline how our vector + GOI is created for Saccharomyces cerevisiae expression system.
Outline how a GFP tag can be used to ensure that our protein is being localized to the correct region of the cell? Why is it useful?
How can we reduce the level of misfolding in our expression system?
Are yeast expression systems perfect for expressing human proteins?
No, it can’t be perfect!
Yeast are still quite different from humans in terms of protein processing –> i.e. PTM –> Results in difficulty in expression of native protein
Can insect cell systems be used to express proteins of interest?
Yup!
Uses recombinant baculovirus which is a virus that is modified with gene of interest and used to infect insect cells - basically using a virus system in insect cells to produce our protein
Robust system, we understand it, not dangerous towards humans.
Bacmid DNA (Virus) contains all the genetic information need to create a virus + our protein of interest
Give a detailed explanation for how insect cell systems are used to express our protein of interest.
- Form recombinant donor plasmid - has a promotor that is going to be used by the Bacmid to produce a protein upon infection (promoter is activated in a particular environment).
- Donor plasmid are transformed into special E. Coli cells containing bacmid DNA - Bacmid contains all the DNA that a virus requires to infect, integrate and kill an insect cell
- Upon transformation we get exchange of donor plasmid with a region in the Bacmid - recombination leads to insertion
- This colonies containing Bacmid are selected for and amplified (antibiotic) –> extraction, amplification and purification
- Transfection into insect cells - leave the cells till they die –> Bacmid takes over cellular system to produce recombinant viral particles
- After 72-96 hours insect cells die resulting in lysis thus releasing the viral particles
- We can assess how much virus is in the media using a viral titter assay - From this information we know how much to add to colony of insect cells to get effective infection
- Take the viral particles and use them to infect insect cells
This time however you leave the cells for one or two days Within this short time period we don’t have complete cellular death yet but we do have production of our protein of interest.
Note - key vocab used –> first round is a transfection (DNA) whereas subsequent rounds are infection (viral particles)
Why don’t we just transfect (insert DNA) the cells instead of using the infection with the Bacmid virus?
Can mammalian cell systems be used for protein expression? Why is it useful?
Yes!
What are some key characteristics of the vector for a mammalian cell system?
Key characteristics of this vector:
- Halo tag - like GFP but we need a substrate to switch on –> expensive
- CMW promoter - Constitutive promoter –> protein will always be produced/promoter is always on.
- MCS –> several restriction sites for insertion
- Two antibiotic resistance genes –> one for screening in E. Coli and the other in mammalian cells
Why E. Coli?
E. Coli is used for cloning and amplification steps
Is there a lot of variation when it comes to vectors in mammalian expression systems?
Are there a lot of different cell types avaliable for mammalian expression systems?
How do we get the DNA into the mammalian cells?
What are the two different types of transfection?
What is a cell free expression system?
Brief overview of the advantages and disadvantages of bacteria expression systems?
Brief overview of the advantages and disadvantages of yeast expression systems?
Brief overview of the advantages and disadvantages of insect expression systems?
Brief overview of the advantages and disadvantages of mammalian expression systems?
Brief overview of the advantages and disadvantages of cell free expression systems?
What is protein engeneering?
Protein engineering - altering proteins at the genetic level in order to change its properties to form an enhanced protein.
All engineering starts with mutagenesis - How do we generate mutants?
Synthetic gene route –> design a new gene –> send it to a company that generate gene for us –> helpful if many substitutions are desired.
Useful applications of protein engineering?
What is the most common way to perform single point mutations?
Must common way to perform a single point mutation/substitution…
Plasmid based approach, Commercially knwon as The Quikchange protocol from Stratagene.
Outline the Quikchange protocol from Stratagene.
The Quikchange protocol from Stratagene, what are some important things to consider when designing a primer?
What is an alternative to the Quickchange method of introducing a single mutation?
Overlap extension methods using PCR also allows us to incorporate a desired mutation
Outline the Overlap extension method used to introduce point mutations.
What is alanine scanning mutagenesis?
Creating many single mutants using Quickchange or Overlap extension methods in order to…
Take a protein and change every A.A to Alanine and if it is already alanine, we change it to Leucine (individually) –> followed by investigating the impacts of the mutation (Perform a functional assay)
This is a systematic approach to engineering proteins - Used to investigate role of residues as well as to obtain a more stable construct.
What does this schematic show us? It corresponds to the β1 adrenergic receptor G-protein coupled receptor
Ligand (A), Receptor (R) and G-protein (G)
Why might we need to engineer this protein?
First step perfomred by researches when attempting to engineer β1 adrenergic receptor?
Second step perfomred by researches when attempting to engineer β1 adrenergic receptor?
Basically, creating mutants at each position and assessing thermostability.
Third step perfomred by researches when attempting to engineer β1 adrenergic receptor?
Testing the ability of the receptor to bind to antagonist and agonist.
Antagonist - Binds and blocks
Agonist - Binds and activates (not substrate)
What is the random mutagenesis technique in protein engineering?
Random mutagenesis - create many mutants and screen for desirable which can then be select from.
What are the different techniques used for random mutagenesis?
Outline how DNA shuffling takes place using LacZ as an example.
Use image to help explain
Principle use homologs from for the same gene that differ slightly –> fragment them and allow them to anneal and amplify using PCR –> creates different combinations
Thus, acting as method for random mutagenesis
Is random mutagensis an iterative process?
After performing random mutagenesis, how do we know which proteins retain function?
How can we combine mutliple mutations to test whether they are beneficial?
Iterative saturation mutagenesis (ISM) for rapid directed evolution
- Start by performing individual mutations at specific sites
- Combine these with mutations at different sites
- Use the best result to perform further mutations until you end up with the desired characteristic
Once again it is an iterative process
Lipase of Bacillus - Iterative saturation mutagenesis (ISM) case study?
Summary of protein engineering?
How does directed mutagenesis/direct evolution take place in the lab?
What protein was engineered in Byrne Lab?
What was the first thing done in Byrne Laboratory when studying uapA (1)?
Hint - Expression
Basically, check whether the S. Cerevisiae expression system produced functional protein that was correctly trafficked
What was the second thing done in Byrne Laboratory when studying uapA (2)?
Hint - Purification
What was the third thing done in Byrne Laboratory when studying uapA (3)?
Hint - Mutants
What was the fourth thing done in Byrne Laboratory when studying uapA (4)?
Hint - Size Exclusion Chromatography
What was the fifth thing done in Byrne Laboratory when studying uapA (5)?
Hint - Long term stability?
What was the sixth thing done in Byrne Laboratory when studying uapA (6)?
Hint - X-Ray crystallography?
What is the fundamental difference between the vector systems of Pichia and S. Cerevisiae?
Vector Systems have been built in different ways!
Pichia Plasmid
HR doesn’t work for generation of the Pichia plasmid as the vectors have been built to work with restriction sites. They do contain HR sites, for integration of the plasmid into the host genome.
S. Cerevisiae
The System contains HR sites that we use for cloning our gene into the vector. The vector does not integrate into the host genome but expression is from the plasmid.
