Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
22 Cards in this Set
- Front
- Back
|
When is mammalian expression used?
|
Where exact post-translational modifications are essential, for example, the addition of sialic acid in the glycans.
|
|
|
What features do mammalian expression vectors have?
|
-Strong viral promoter for expression
-Use of a viral origin of replication e.g. SV40, and selectable marker -Use of an E.coli origin and selectable marker Addition of signals for secretion and addition of fusion proteins. |
|
|
What was the first virus promoter used in mammalian expression and what did it need?
|
Simian virus 40 (SV40); needed transcription enhancers, promoter, splicing signal and a polyadenylation signal
|
|
|
What similar elements to those of SV40 from other viruses are used?
|
Cytomegalovirus (CMV)
Rous sarcoma virus (RSV) Semlili forest virus (SRV) |
|
|
What are common selectable markers used in mammalian expression systems?
|
Neo gene that encodes resistance to neomycin
DHFR gene that confers resistance to methotrexate |
|
|
What importance does dihydrofolate reductase (DHFR) have in the cell and how can we exploit it?
|
It reduces dihydrofolate to tetrahydrofolate which is essential for purine synthesis. MTX binds in the active site of DHFR, acting as a competitor to kill cells. Only cells which increase number of DHFR enzymes survive; if we put recombinant gene on vector for DHFR then yield will increase with increase in DHFR when we increase MTX conc.
|
|
|
What cells do we use for transient expression?
|
Green monkey kidney cells
Baby hamster kidney cells Human embryonic kidney cells |
|
|
How is DNA introduced into the mammalian cell cytosol?
|
Microinjection
Electroporation Calcium phosphate Cationic lipids Liposome-mediated Viral vector |
|
|
What do you add to get DNA to nucleus to be transcribed?
|
Nuclear targeting sequences
|
|
|
What are the advantages and disadvantages of creating a stable cell line?
|
This is where the plasmid has been transfected into the cell line and then incorporated into the genome.
Advantages: long term production Disadvantages: time-consuming, costly |
|
|
Give an example of a recombinant protein made in mammalian cells to treat patients with cystic fibrosis.
|
Human DNase I
37kDa secreted glycoprotein with 2 N-glycosylation sites and 2 disulphide bonds. Contains sialic acid in the glycans. First expressed in CHO cells in 1990 by Genentech scientists- now sold as Pulmozyme |
|
|
How was DNase I specially designed to stop actin inhibition of it in the lungs?
|
Site-directed mutagenesis. They also tried to engineer in another glycosylation site near the actin binding sit to sterically inhibit actin binding
|
|
|
What is Factor VIII?
|
Blood clotting factor used to treat patients with haemophilia A. Made in CHO cells by Bayer but very expensive to synthesise and purify because it's very big with lots of recombination events (25 positions need to be N-link glycosylated and needs some aas sulphated and o-glycosylated)
|
|
|
How did they increase the efficacy of Factor VIII?
|
Increase efficiency of activation
Increase resistance to inactivation Decrease antigenicity |
|
|
How can cell-free expression systems be used?
|
To synthesise proteins in vitro. Uses extracts from a cell containing all the machinery and biochemical constituents required for transcription and translation.
|
|
|
What is the rapid translation system?
|
Made by Roche from an E.coli lysate, it prevents toxicity problems and makes membrane protein expression easier by allowing for the replenishment of important components of the reaction which are being consumed via diffusion.
|
|
|
What are the advantages of E.coli lysates?
|
Easy to prepare an extract from E.coli
Improved to increase time that system will work in vitro Yields for some proteins are more than 1mg/ml Additional factors can be added to help syntehesise certain proteins |
|
|
How did scientists induce the production of eukaryotic protein with multiple disulfides and a multimeric cofactor-containing protein?
|
Added a range of E.coli chaperones and the co-factor directly into the reaction mix
|
|
|
What is a wheat germ cell-free system?
|
Extracting stored components from a wheat germ embryo and using them to synthesis a protein for up to 60h
|
|
|
What is the advantage of being able to PCR amplify any cDNA to contain the correct 5' and 3' UTRS in wheat germ?
|
PCR product alone can be used in the system
We can use it in continuous exchange system There doesn't have to be a strong codon preference Wheat seeds are cheap and safe Easy to scale up for high-throughput protein pipelines. Can produce 100s of proteins a day |
|
|
What is the disadvantage of the wheat germ cell-free system?
|
Doesn't carry out glyosylation
|
|
|
How might we mimic the membrane environment -and therefore enable glycosylation- in cell-free expression systems?
|
Use protein nanodisks
Liposome Add detergent, such as DMM, to allow the membrane protein to directly insert into detergent micelles. |
