Question

Combined chromatography methods

You are provided with a solution containing a mixture of three different proteins with the following characteristics.

Protein	mol. mass (Da)	isoelectric point (pI)	Binds to ligand?
A	45,000	6.1	Yes
B	84,000	7.8	No
C	85,000	7.9	Yes
D	120,000	6	No
E	122,000	8.0	No

You are also provided with a size exclusion column, an ion-exchange column, and an affinity purification column (containing the ligand recognised by proteins A and C above).

With the information above, how might you go about purifying:

Protein A from the combined mixture?

Size exclusion

Ion-exchange

Hydrophobic interaction chromatography

Affinity purification

Protein D from the combined mixture?

Size exclusion

Ion-exchange

Ion-exchange followed by size exclusion

Affinity purification followed by size exclusion

Protein C from the combined mixture?

Size exclusion

Affinity purification

Ion-exchange followed by size exclusion

Affinity purification followed by size exclusion

Answer 1

Purifying protein A from the combined mixture

If we compare the molecular mass of all the proteins, protein A is having the least molecular mass. The molecular mass of other proteins are nowhere closer to that of protein A. Therefore, the best way to purify protein A is through size exclusion chromatography.

Ion exchange cannot be used because the isoelectric point of protein A and protein D are closer which means that they have similar charge.

Hydrophobic interaction chromatography is best suited for intermediate purification and polishing steps. Hence it is not the best method to be used here.

Affinity purification cannot be done because the ligand of the affinity purification column (in this case) can bind to both protein A and protein C.

Purifying protein D from the combined mixture

Comparing the molecular masses and the isoelectric points the best way to purify protein D is through ion exchange followed by size exclusion. When we do ion exchange, a mixture protein D and protein A are purified because of their close isoelectric points (similar charge). However, there is a huge difference in the molecular masses of these proteins. Hence, size exclusion can separate protein D from protein A.

Size exclusion alone cannot be used because the molecular mass of protein D and E are closer.

Ion exchange alone cannot be used because the isoelectric point of protein D and A are closer.

Affinity purification (followed by size exclusion) cannot be done because protein D does not bind to ligand.

Purifying protein C from the combined mixture

Comparing the molecular masses and the isoelectric points the best way to purify protein C is through affinity purification followed by size exclusion. Since the ligand of the affinity purification column can recognize and bind to both protein C and protein A, a mixture of these proteins are purified. However, the differences in their molecular masses can be exploited to purify protein C from the purified mixture of protein A and C with size exclusion.

Size exclusion alone cannot be used because the molecular mass of protein B and C are closer.

Affinity purification alone cannot be done because the affinity purification column can recognize and bind to both protein C and protein A.

Ion exchange followed by size exclusion cannot be used because both isoelectric points and molecular mass are closer for protein B and C.