Question

Say you have a mixture of four proteins with the following properties:

Protein A: MW of 30 kDa, pI = 5.0, binds to nitrilotriacetic acid

Protein B: MW of 35 kDa, pI = 4.5, low solubility and will precipitate in 5% (w/v) (NH4)2SO4

Protein C: MW of 100 kDa, pI = 4.7

Protein D: MW of 30 kDa, pI = 7.4

(a) Using a flow chart, propose a purification strategy to purify and separate the proteins

(b) What technique(s) would you use to verify the success of each step of your purification?

Answer 1

ANSWER A and B :-

1. (Isolation of Protein D) The first method that should be made applicable is isoelectric focussing of the protein molecules. The pH should be kept at 6 and this would result in the separation of two types of proteins namely one which will be positively chared and the others which will be negatively charged. The Protein D will be positively charged mainly because it has a pI value which is more shifted towards the basic value as compared to the pH. The other remaining proteins will be negatively charged as their pI is low than the pH of the solution in which separation is being carried out.
2. (Isolation of Protein C) The next method that should be included is the size exclusion chromatography wherein the molecules are being separated on the basis of their size. From the remaining molecules, Protein C seems to have a relatively bigger molecular weight as compared to the remaining two proteins. This would be separated first taking into consideration the size of the beads which are being used in order to mediate the separation. This is followed by elution of the remaining components.
3. (Isolation of Protein A) The next method should be separation carried out by means of Nitrilotriacetic acid which shows affinity towards the molecule such as Protein A such a way that the remaining components will be separated.
4. The last step will be the isolation of Protein B using (NH4)2SO4 which will also act as a confirmation test for the detection of protein B as it shows the lowest solubility to be separated forming a precipitate.
5. According to me, the process of isoelectric focussing is very accurate and provides a positive result as compared to other methods of protein purification. But, if the accuracy of whether the protein has been purified needs to be checked, specific interactions can be carried out as in case of immunoblotting to identify whether the desired protein is specific in terms of its interaction and can thus highlight any impurity if present.
6. Gel electrophoresis technique can be used to check the efficiency of separation of the Protein C as it tends to have a relatively larger molecular weight.
7. The separation carried out by using Nitrilotriacetic acid can be evaluated in certain ways out of which one is centrifugation to separate the target protein A.