Protein Purification

Question 1

Main steps protein purification

Answer 1

Break cells
Separate proteins by running chromatography
Solution of isolated protein

Question 2

Protein solubility in Bacteria (Solubility Tags)

Answer 2

Highly soluble proteins that ensures tagged proteins are soluble
Can be at N or C terminus
Can have mulltiple tags

Question 3

Ex. Solubility tags

Answer 3

His tag
Maltose Binding Protein (MBP) tag
Glutathione S transferase (GST) tag

Question 4

Twto typea of chromatography for protein purifications

Answer 4

Affinity based Chromatography
Size exclusion Chromatography

Question 5

Affinity based Chromatography

Answer 5

Matriz that binds to solubility tag
- Nickel matrix for His tag
- MPB matrix for MBP tag
- GST matrix for GST tag

Target protein eluted w/ competitive ligand
- Nickel: imidazole
- MBP: maltose
- GST: glutathione

Question 6

Size exclusion chromatography

Answer 6

Separate based on size & shape:
- Larger preoteins elutes first
- Smaller proteins travel through beads