Purifying Protein

Question 1

Why should we recombinantly express proteins?

Answer 1

1. to increase yield
   - more protein per starting material
2. ease of purification
3. labelling
   - isotopic labelling
4. ethical considerations
5. genetic modifications
   - test/ alter functions

Question 2

What is recombinantly expressing proteins?

Answer 2

Putting a gene into something in order for it to make that protein

Question 3

What are some properties of vectors?

Answer 3

• origin of replication
• selectivity marker
• for transcription sequences
• protein codons must be in reading frame
• inducible promoter (can tell organism when to make protein)

Question 4

What are the different hosts that can be used?

Answer 4

• bacteria
• yeast
• insect
• mammalian

Question 5

What are the pros and cons of using bacteria as a host?

Answer 5

pros:
- cheap
- fast
- high yield
cons:
- struggle with eukaryotic proteins
- can cause inclusion bodies

Question 6

What are the pros and cons of using yeast as a host?

Answer 6

pros:
- cheap
- fast
- mid-range yield
cons:
- different glycosylation pattern from higher eukaryotes

Question 7

What are the pros and cons of using insect cells as a host?

Answer 7

pros:
- better for eukaryotic proteins
- often good yield
cons:
- slow
- reasonably expensive

Question 8

What are the pros and cons of using mammalian cells as a host?

Answer 8

pros:
- good for eukaryotic proteins
cons:
- slow
- expensive
- poor yield

Question 9

What are the different purification approaches?

Answer 9

• cell press
• sonication
• centrifugation
• precipitation
• solubilisation (polymer that will chop our membrane proteins and keep them with its native lipids in vivo)
• dialysis

Question 10

What are the different column chromatography methods?

Answer 10

• size exclusion
• ion exchange
• affinity methods

Question 11

What happens during ion-exchange chromatography?

Answer 11

• separation by ionisation potential (how much they stick to the resin in the column)

Question 12

What happens during gel-filtration chromatography?

Answer 12

• separation by size
• porous beads will restrict
• large molecules will come out first
• smaller molecules will come out last

Question 13

What happens during affinity chromatography?

Answer 13

• beads with covalently attached substrate

- enzyme molecules will pass through and bind

Question 14

How do the Ni2+/Co2+ affinity columns make use of the poly-histine tag?

Answer 14

• the histidine will stick to the Ni and Co in the column
• rest of the proteins will be passed on and removed
• the proteins with Ni and Co can be removed after with competing ions

Question 15

What factors affect the choice of affinity-tag?

Answer 15

Size:
- :) small tags are less like to affect folding/ activity
- :( can become obscured so no binding
Stability:
- a larger tag can increase stability of the protein