Question

What fractionation procedure could be used to purify protein 1 from a mixture of three proteins whose amino acid compositions are as follows?

1. 25% Ala, 20% Gly, 20% Ser, 10% Ile, 10% Val, 5% Asn, 5% Gln, 5% Pro

2. 30% Gln, 25% Glu, 20% Lys, 15% Ser, 10% Cys

3. 25% Asn, 20% Gly, 20% Asp, 20% Ser, 10% Lys, 5% Tyr

All three proteins are similar in size and pI, and there is no antibody available for protein 1.

Short answer

Ion Exchange Chromatography can be used to purify protein 1 from the mixture of given three proteins.

Step-by-step solution

Ion Exchange Chromatography

Ion Exchange Chromatography is a fractionation process used to separate charged molecules (anions and cations). In this process, charged molecules attach to oppositely charged groups which are chemically bound to a matrix such as cellulose or agarose.

On anion exchangers, anions bind to cationic groups, while on cation exchangers, cations bind to anionic groups. A matrix with linked diethylaminomethyl (DEAE) groups is perhaps the most commonly used anion exchanger, whereas a matrix with attached carboxymethyl (CM) groups is probably the most commonly used cation exchanger.

Process of Ion Exchange Chromatography

The proteins to be extracted are dissolved in a buffer with proper pH and salt concentration before being transferred to an ion exchanger column. The buffer is then used to wash the column. Proteins with low affinities for the ion exchanger pass through the column faster than proteins with higher affinities as the column is washed. The column effluent is then collected in a number of fractions. Proteins that bind tightly to the column's ion exchanger can be eluted (washed through the column) by using an eluant (a buffer with a greater salt concentration or a pH that lowers the matrix's affinity for the protein).

Fig: Purification by Ion Exchange Chromatography

Explanation

When the mixture containing the 3 proteins is passed through an ion exchanger column containing an anion exchanger, protein 2 and protein 3 will bind to these anion exchangers. Protein 2 and protein 3 contain aspartic acid and glutamic acid which are negatively charged amino acids. Protein 1 will pass through the column at a faster rate and will be eluted out of the column. Protein 2 and protein 3 can be eluted by using an eluant. In this way we can separate protein 1 from the mixture of given 3 proteins.