Question

A DNA-binding protein is needed to be expressed and purified for structural studies. It has the following properties: (a) It is a human protein;

(b) The overall molecular mass is about 150 kDa;

(c) It contains rare codons;

(d) It has multiple charged and hydrophobic amino acid residues;

(e) Its pI is 5.4;

(f) It is sensitive to proteolytic degradation.

Starting from the design for protein expression using an appropriate expression system, briefly describe how you would proceed logically to purify this protein to homogeneity (pure form). You may describe your answer with the help of a flow-chart if necessary.

Answer 1

Gel filtration is used for separation of protein and other biomolecules and also used for determination of its molecular weight. It is based on the stokes radius. Stokes radius is the radius of hypothetical sphare formed by trumbling of biomolecule in the solution. Stokes radious is influenced by shape and size of molecule.

Protein exist in three conformation namely called native, molten and denature state. Gel filtration chromatography denature protein illuted before the molten globule which is illuted before the native protein.

Gel filtration chromatography can be used to measure molecular size, to isolate components in a mixture ot to extract salt or exchange buffers from a macromolecular preparation.

G100 or G 200 can be used for such protein purification.