Practical Aspects

Question 1

Give 3 advantages of E. Coli as an expression system.

Answer 1

Many vectors & strains optimised for expression
Quick if works
Expression levels can be high & inducible
Safe category I (bench)

Question 2

Give 2 disadvantages of E. Coli as an expression system.

Answer 2

No PTM

Cell reducing so disulphide bonds forms incorrectly –> inclusion bodies if protein has lots of disulphides.

Question 3

Why might you use baculoviruses?

Answer 3

nonpathogenic (can’t replicate in mammalian cells)
Insect cells can be used which post-translationally modify proteins similarly to mammalian cells.
Good for crystallography as homogeneously glycosylated.

Question 4

Why no to baculovirus?

Answer 4

maintain viral stock (not indefinite & only for ~3mths)
Sensitive to environmental changes
Time consuming expression & expensive.

Question 5

Why is yeast not completely advantageous?

Answer 5

glycosylation less complex than mammalian cells (may affect activity & structure)

Question 6

What are the advantages of Mammalian expression systems?

Answer 6

PTMs

Permanent cell lines express high protein levels (1–mg/L)

Question 7

Why are mammalian expression systems difficult?

Answer 7

time consuming to make permanent high expressing line.
Less safe than E. Coli (active protein made)
Expensive (specialist equipment & contamination issues)

Question 8

what three ways can oligo’s attack proteins?

Answer 8

• N glycosidic bond to R group of Asn in consensus Asn-X-Ser/Thr
• O glycosidic (R group pf Ser or Thr) - extensive for structural ps.
• GPI anchor component used to attach proteins to CM.

Question 9

What is the Kozak sequence?

Answer 9

RccAUGG, where R is A or G 3 bases upstream from AUG start codon.
Initiates translation
Strength of Kozak sequence can affect expression levels.