Expression Of Recombinant In Eukaryotic

Question 0

What post translational modifications do eukaryotic systems allow to occur?

Answer 0

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

Question 1

What are the main problems with prokaryotic expression systems?

Answer 1

Incorrect protein folding
Protein insolubility
Incorrect post-translational modification

Question 2

What is a shuttle vector?

Answer 2

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

Question 3

What are plasmid cloning vectors modified to include?

Answer 3

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

Modifications needed to cross the Prokaryotic/eukaryotic boundary.

Question 4

What are the six key components of a eukaryotic vector?

Answer 4

Many eager priests touch infant boys
BTIPEM
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

Question 5

What’s the main advantage of viruses?

Answer 5

High rates of infection

Question 6

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

Answer 6

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

Question 7

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

Answer 7

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

Question 8

What is lipofection? What is it particularly useful for?

Answer 8

Liposome mediated gene transfer

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

Question 9

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

Answer 9

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

Question 10

What are selectable markers? What are they developed for?

Answer 10

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

Developed for eukaryotic cells to allow creation of stable cell lines

Question 11

What is GFP

Answer 11

Green fluorescent protein

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

Question 12

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

Answer 12

• 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)

Question 13

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

Answer 13

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

Question 14

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

Answer 14

Two vectors commonly produced in yeast

Question 15

What are the advantages of using baculovirus vectors?

Answer 15

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

Question 16

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

Answer 16

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

Question 17

Draw and label a baculovirus transfer vector

Answer 17

Include

• 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)

Question 18

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

Answer 18

As per mammalian gene expression slide 12

Question 19

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

Answer 19

As per mammalian gene expression slide 12

Question 20

What are the different methods available to transfect eukaryotic cells?

Answer 20

Yeasts and shuttle vectors

Insect cells and baculovirus systems