Question

1. A biochemist is attempting to separate a DNA-binding protein (protein X) from other proteins in a solution. Only three other proteins (A, B, and C) are present. The proteins have the following properties: pl (isoelectric point) Size Mr Bind to DNA? protein A 7.4 protein B 3.8 protein C 7.9 protein X7.8 82,000 21,500 23,000 22,000 yes yes no yes What type of protein separation techniques might she use to protein X from the other proteins. Give a flow chart and explain why you choose each technique. (10 points) 2. As a protein is purified, both the amount of total protein and the activity of the purified protein decrease. Why, then, does the specific activity of the purified protein increase? (5 points)

Answer 1

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ANS::

Protein A has a pi of 7.4 size of 82000 and it does bind to DNA. Protein X has a pi of 7.8 size of 22000 and it also binds to DNA. So here the best way to separate them is to apply the separation process of SDS-PAGE analysis that is Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis which separates the proteins based on their molecular weight or size.

Protein B and Protein X has almost simillar size so SDS-PAGE technique would not be effective in their separation. Also both are DNA binding proteins. But they differ in their pi. Protein B has a pi of 3.8 and Protein X has a pi of 7.8. So we apply the technique of isoelectric focussing. We setup a pH gradient by allowing a mixture of organic acids and bases. Proteins migrate upto the pH which equalls their pi. At pH = pi the net charge on the protein is zero so they do not migrate further.

Protein C has a pi of 7.9 and size of 23000 while protein X has a pi of 7.8 and a size of 22000. That is they have almost simillar pi and size. So applying SDS-PAGE or isoelectric focussing technique will not be effective in their separation. Protein C does not bind to DNA but protein X binds to DNA. So in this case we apply the DNA affinity chromatography technique which is based on binding specificities. The proteins which bind to DNA are retained on the column while those which do not bind are washed through to column.