Expression

Question 1

Why is e.coli used as a system?

Answer 1

Lots of info about it

Range of strains

Lots of vectors (regulated expression)

Large numbers produced cheaply.

Question 2

What is the codon usage problem?

Answer 2

Relative occurrence of codons for same AAs may vary between species

• mutate codons
• modify bacteria to express more tRNAs

Question 3

How can you select for eukaryotic cells that take up vectors?

Answer 3

Neomycin (inhibitor of eukaryotic translation)

Blasticidin S

Question 4

When inserting vectors into eukaryotic cells what is important?

Answer 4

To use a promoter active in eukaryotes such as CMV

Question 5

What do pGEX vectors do?

Answer 5

Allows cDNAs to be recombinantly expressed as GST fusions.

Question 6

What is the elation step for GST fusions?

Answer 6

Add glutathione alone to column.

Question 7

What are the main issues associated with fusion peptide?

Answer 7

They may be large (15-20 kDa) prevent correct folding altering properties of RP.

Could remove fusion.