Question

List the types of protein purification techniques we discussed. State what chemical or physical property of the protein they work on. Describe a three-step purification procedure of a protein with a mass of 150 kDa, high hydrophobicity, and a pI of 9.5 from a mixture of many proteins. Give reasoning for your choice of each step of the procedure.

Answer 1

Protein purification includes the following steps:

1. Extraction of protein : the purpose of protein extraction is to increase the quality of protein content in the cell lysate. Extraction of proteins can be done by sonication, homogenisation under high pressure or by use of detergents.

2. Precipitation of the extracted protein : extracted proteins are precipitate with the help of ammonium sulphate.

3. protein purification techniques:

A. Gel filtration chromatography: separation on the basis of molecular weight and size of the molecule, molecules with high molecular weight and larger size elute out first from the column and smaller molecules elute out at last.

B. Ion exchange chromatography: separation on the basis of charge on molecule or isoelectric point.

C. Affinity chromatography : separates molecules based on reversible interactions based on biological recognition. Eg. Antigen-antibody interactions.

D. High pressure liquid chromatography : separates molecules on the basis of hydrophobicity of molecules, less hydrophobic molecules elute out first from the column.

E. Chromatofocusing : Purifies protein on the basis of isoelectric point.

A protein of mass 150 KDa, high hydrophobicity and pI of 9.5 can be separated from a mixture of proteins by following steps

• Gel filtration/size exclusion chromatography (it will separate the protein on basis of molecular weight).
• HPLC, this will yield more purified form of protein on the basis of hydrophobicity.
• Finally isoelectric focusing can be done, as two proteins having same molecular weight and hydrophobicity of the sample protein will have isoelectric point different from it, hence sample protein having pI of 9.5 will be separated.