Bernadette Byrne Flashcards

Question

What are some examples of commonly used tags? What are they used for?

Answer 1

Is the protein present? --\> can also be figured out by using a western blot - uses antibodies for particular protein

Answer 2

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.**

Answer 3

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.

Answer 4

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

Answer 5

These are the most common: 1. Yeast - Pichia or Saccharomyces cerevisiae 2. Insect - Viral based infection 3. Mammalian - Transient or stable 4. Cell free

Answer 6

1. 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 2. Specific protein requirements - molecules, cofactors, chaperones, etc. 3. What is the downstream application? - what are you going to do with the protein --\> structural studies? Examining behavior of protein in cell? Etc.

Answer 7

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.

Answer 8

**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

Answer 9

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

Answer 10

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

Answer 11

1. 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). 2. 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 3. Upon transformation we get exchange of donor plasmid with a region in the Bacmid - recombination leads to insertion 4. This colonies containing Bacmid are selected for and amplified (antibiotic) --\> extraction, amplification and purification 5. Transfection into insect cells - leave the cells till they die --\> Bacmid takes over cellular system to produce recombinant viral particles 6. After 72-96 hours insect cells die resulting in lysis thus releasing the viral particles 7. 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 8. 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)

Answer 12

Key characteristics of this vector: 1. Halo tag - like GFP but we need a substrate to switch on --\> expensive 2. CMW promoter - Constitutive promoter --\> protein will always be produced/promoter is always on. 3. MCS --\> several restriction sites for insertion 4. 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

Answer 13

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.

Answer 14

Must common way to perform a single point mutation/substitution... Plasmid based approach, Commercially knwon as The Quikchange protocol from Stratagene.

Answer 15

Overlap extension methods using PCR also allows us to incorporate a desired mutation

Answer 16

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.

Answer 17

Basically, creating mutants at each position and assessing thermostability.

Answer 18

Testing the ability of the receptor to bind to antagonist and agonist. Antagonist - Binds and blocks Agonist - Binds and activates (not substrate)

Answer 19

Random mutagenesis - create many mutants and screen for desirable which can then be select from.

Answer 20

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

Answer 21

**Iterative saturation mutagenesis (ISM)** for rapid directed evolution 1. Start by performing individual mutations at specific sites 2. Combine these with mutations at different sites 3. Use the best result to perform further mutations until you end up with the desired characteristic Once again it is an iterative process

Answer 22

Basically, check whether the S. Cerevisiae expression system produced functional protein that was correctly trafficked

Answer 23

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.