Question 2. You have managed to express your his-tagged protein --Congratulations! [Total = 11 marks].

a. Which chromatography technique will you use to purify this His-tagged protein? [1 mark]

Answer:

b. Please explain to your supervisor how you will do it? (what are the steps in this technique?) [3 marks].

Answer:

c. Unfortunately, when you run your protein on a gel, you see that a set of very large proteins co-purified with

your His-tagged HGH (see picture below). You want to get rid of this very large protein. Which

chromatography technique do you suggest to your supervisor can be used to solve this problem [1 mark]?

Answer:

250

100

10

Contaminant proteins (>250 kDa)

His-tagged HGH (22kDa)

d. Explain the principle of the technique to your supervisor [3 marks].

Answer:

e. You have now a purified protein - Well done! You assess its specific activity using the table below (testing

the activity of your protein on a mammalian cell proliferation assay).

Step

Volume (ml)

Crude bacterial

extracts

After your first

step (Question 4a)

After you second

step (Question 4b)

500

1

1

Total protein (mg) Total HGH activity

(units)

20,000

10

5

1,000

900

800

Specific activity

(units/mg of

protein)

0.05

i) What is the specific activity of your purified protein (bottom right cell in the Table)? [1 mark].

Answer:

90

ii) Using the numbers in this table, by which factor have you purified the specific activity of your protein between

the start (crude extract) and the final step? [1 mark].

Answer:

iii) How much (in mg) of the large, contaminating protein in the figure above did you remove by doing your second

step? [1 mark].

Answer: