Mirco Lab 2 Midterm: Lecture 4

Question 1

The benefits of making fusion proteins

Answer 1

Convenient for isolation and purification of
the protein

• Convenient for detection
• May direct the protein to specific cellular
  compartment
• May protect from proteolysis
• May improve solubility

Question 2

Describe the cleavage of the tags

Answer 2

Enzymatic cleavage by specific proteases at sites
incorporated in the vector between the fusion protein
and the cloned protein

• Factor Xa: IE(D)GR (pMAL, pGEX)
• αThrombin: LVPRGS (pGEX 2T)
• TEV protease: ENLYFQG (pRoEX)
• Enterokinase: DDDDK
• Potential problems: does not work efficiently, it might
  work better with denatured proteins, presence of
  cleavage sites in the cloned protein

Question 3

Fusion proteins solubility

Answer 3

Size of the expressed protein

• The nature of the expressed protein: TF,
  enzyme,

transmembrane protein

• Expression level
• Expression system
• Nature of the fusion tag