Perfect and problematic expression

Question 1

Why is there a protein solubility problem in E. coli?

Answer 1

The dissimilarity of eukaryotic proteins and E. coli post-translational processes

Question 2

What can’t E. coli do to a protein?

Answer 2

Glycosylation

Question 3

What happens to the proteins produced?

Answer 3

They are produced as insoluble deposits in the cell

Question 4

Describe denaturation:renaturation

Answer 4

Add denaturants to form linear polypeptides

Remove denaturants to refold into soluble proteins

Question 5

How to avoid the protein folding problem?

Answer 5

Express the protein at a lower temperature
Express the protein at a lower level to avoid the aggregate formation
Target the protein to the periplasm as folding and disulphide interchange occurs more readily in this compartment
Express protein as a fusion with GST or thioredoxin to enhance disulphide bond interchange and folding in the cytoplasm
Use host strain for expression which overexpresses chaperonins
Use eukaryotic host instead