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Flashcards in Expression Of Recombinant In Eukaryotic Deck (21)
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What are the main problems with prokaryotic expression systems?

Incorrect protein folding
Protein insolubility
Incorrect post-translational modification


What post translational modifications do eukaryotic systems allow to occur?

Correct disulfide bonds
Proteolytic cleavage of inactive precursor
Phosphorylation, sulfatation, acetylation, prenylation


What is a shuttle vector?

A cloning vector capable of replicating in two or more types of organism


What are plasmid cloning vectors modified to include?

Promoter and transcription terminator if needed (suitable to the host organism)

Modifications needed to cross the Prokaryotic/eukaryotic boundary.


What are the six key components of a eukaryotic vector?

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1) backbone shuttle sequences for providing replication
2) translational initiation sequence (appropriate)
3) intron
4) polyadenylation signal (for mammalian vectors)
5) marker gene for selection


What's the main advantage of viruses?

High rates of infection


What are the four different promoters and what are they used for?

1) minimal promoters, used to study gene regulatory elements (eg enhancers)
2) constitutive promoters, used to direct expression of gene products
3) cell specific promoters, used to specify expression to tArget cells
4) regulated promoters, used to control the on/off expression of cloned genes


What are some methods of introducing recombinant DNA into host cells?

- calcium chloride and heat shock
- micro injection
- calcium phosphate precipitation
- lipofection
- electroporation
- nucleofection
- viral infection
- nanoparticles


What is lipofection? What is it particularly useful for?

Liposome mediated gene transfer

Useful for large DNA molecules since the lipid membrane protects them from damage by shearing forces


How does electroporation work (in getting DNA to enter cells)

Cells and DNA are mixed in chambers and an electrical pulse is passed through
This temporarily opens holes allowing DNA to enter cells

This must be optimised to each cell type to find conditions where take up is maximal and cell death is minimal


What are selectable markers? What are they developed for?

Genes that add a new trait to cells, eg antibiotic and GFP

Developed for eukaryotic cells to allow creation of stable cell lines


What is GFP

Green fluorescent protein
Extremely stable protein of 238 amino acids.... Ultraviolet light causes it to autofluoresce in a bright green colour


What are some advantages of using yeasts to produce recombinant gene products in non mammalian eukaryotic cells?

- simple and easy to manipulate in euk cells
- will give better processing of mammalian cells than bacteria
- gives some post translational modifications but not all (not gamma-carboxylation)


Draw an example of a yeast shuttle vector and fully label the components it needs to allow propagation in both yeast and E. coli cells

Yeast components - leu-2nd needed for leucine synthesis, ori y, tpi promoter and terminator around a restriction site

E. coli - ori B, ampicillin resistance gene


When recombinant human insulin is produced, what is needed to produce the 2 chains?

Two vectors commonly produced in yeast


What are the advantages of using baculovirus vectors?

- recognises most mammalian targeting sequences and can perform many of the post-translational modification performed in mammalian cells
- very large DNA inserts


What is the problem with baculovirus for introducing DNA into a cell?
What is done about it?

Too big to insert DNA directly (150kbp)
- it is first cloned into a transfer vector, and then introduced into the cell by homologous recombination inside host cell


Draw and label a baculovirus transfer vector

- baculovirus flanking sequences to promote recombination, one with a polyhedron promoter and one with a n-terminus
- MHS site at the terminus
- ampr
- ori b (both from E. coli)


Produce a table of the advantages and disadvantages of the yeast expression system

As per mammalian gene expression slide 12


Produce a table of the advantages and disadvantages of an insect expression system

As per mammalian gene expression slide 12


What are the different methods available to transfect eukaryotic cells?

Yeasts and shuttle vectors
Insect cells and baculovirus systems