Question

Protein purification techniques take advantage of the ways that proteins differ from each other with respect to various physical or chemical properties. List five properties of proteins that can be used as basis for their separation and describe a method of separation based on each of these properties. Please explain the physical and/or chemical processes involved for each of the separation techniques you describe.

Answer 1

The five physical and/or chemical property of protein by which proteins can be purified are:-

1. Affinity

2. Size/Shape

3. Charge

4. Hydrophobicity

5. Precipitation

The technique for purifying protein based on each of its physical and/or chemical property is as follows:-

1. Affinity: also can be called as biorecognition, the technique used here is affinity chromatograhy. A protein (for eg, enzyme) is intriduced into a coloumn containing specific substrate to the target protein, this enzyme/ protein binds to the substrate in the gel column and all the other componenets are eluted out, and later to elute the target protein, excess amount of more specific elutent to the protein is added and the protein is retrived.

2. Size/shape:- Size exclusion chromatography is used in separation of protein based on their size, when a hetergenous protein mixture is added to pass thorough the sol-gel column, the gel molecule with a specific size pore will allow smaller molecule to pass thorugh and the larger protein molecule will be stuck inside, this protein (target protein for example), will then be eluted out using excess amount of elutent.

3. Chargre:- To separate protein based on their polarity/charge the ion exhcnage chromatography technique is used, however in cae of protein an isoelectric point method is also to be used jsut after chromatogrphy. In ion exchange chromatography the protein is passed through the sol gel containing the oppositely charged silica beads than the protein. (if positively charged protein is to be separated then silica gel must be negatively charged), so the positively charged protein will bind to the gel and remain in the column and the negatively charged protein will elute out first, then a higher negatively charged elutent is added from the top to elute out the postively charged protein.

4. Hydrophobicity:- Hydrophobic Interaction Chromatography (HIC) is sued. In this technique the protein molecule is separated based on the hydrophobic property of amino acid chain. The HIC column is covered with high salt concentration which generates a pull for the hydrophobic part of the protein, the higher the hydrophobicity, the lower salt concentration is required, and the other step remains complementary to the size exclusion and ion exchange chromatography.

5. Precipitation:- In a process of separating or purifying protein from a complex mixture of other conpounds, precipitation comes as handy technique, where the precipitating property of protein in a high salt concentration helps to separate out the other contaminants, usually ammonium sulphate is used. Higher concentration of salt in the solution precipitates the protein and makes it easier to separate them, The precipitated protein is then separated as fraction and the sal (ammonium sulphate) is removed via dialysis.