Protein Concentration“salting in” and “salting out

Question 1

what’s the first step in protein purification

Answer 1

Extraction and clarification•The first step in any (tissue/cells or recombinant cells) protein purification protocol is the extraction. •There are many methods e.g. mortar and pestle, blender, enzymes (for bacteria) etc.•The cloudy extract will contain unbroken cells, debris etc so the extract must be clarified before applying the extract to a chromatography column (block the pores in the resin)•Centrifugation or ultra filtration are among the many techniques used to clarify an extrac

Question 2

Why concentrate?At the start of a purification

Answer 2

Proteins in the cell normally at high concentrations (50-200mg ml-1) and in reducing conditions.
After extraction the conditions are generally low protein concentration in an oxidising environment.
At the initial stages of purification water can be viewed as a major contaminant. In industry pumping large volumes of dilute feed stock is very expensive
Concentrating the protein solution helps to reduce the volume and may remove inhibitors e.g. metal ions

Question 3

techniques to concentrate protein sample

Answer 3

Salt precipitation (Ammonium Sulphate)
Solvent precipitation (Acetone)
Ultrafiltration (stirred cell, centrifugal cells or tangential flow)
Chromatography (IEX, HIC)

Question 4

what happens to proteins in solution

Answer 4

A solvation shell of water molecules surrounds proteins in solution.A shell of water molecules
2-3 deep surround 
proteins in solution.
The water molecules interact
with polar functional groups 
of amino acids

Question 5

Solubilising properties of water and the salting out of proteins

Answer 5

A solvation shell surrounds proteins interacting with the weak acid groups of proteins.
High salt strips the solvation shell away which exposes patches of hydrophobic amino acids near to the surface of the proteins structure
This is thermodynamically unfavourable. To address this the exposed areas associate to lower the free energy of water.
When the clumps are large enough they precipitate

Question 6

How does water react with hydrophobic molecules

Answer 6

Water forms clathrate “caged” structures around hydrophobic molecules. Clathrate structures are thermodynamically unfavourable

Question 7

Ammonium sulphate in salt precipitation

Answer 7

Ammonium sulphate ppt is generally a very mild method of protein concentration.
Proteins will refold into the correct conformation with little loss of activity
Can store protein as a pellet at -25OC indefinitely.
Only used in lab as ammonium sulphate will corrode stainless steel used in the biotech industry. Also problems with disposal
Works best at a protein concentrations above 1.0mg ml-1

Question 8

The Hofmeister series

Answer 8

The Hofmeister series is a classification of ions in order of their ability to salt out or salt in proteins. The more strongly hydrated anions and weakly hydrated cations cause increasing salting out of proteins. The more weakly hydrated anions and strongly hydrated cations cause increased salting in of proteins.