INTRODUCTION

Widely use in Downstream Processing for

purification purposes.

It is better than dialysis or desalting methods as it

enables concentration of the protein samples as well
as purification from undesired substrates.
MECHANISM OF
PRECIPITATION
It is to alter the solvation potential of the solvent and
thus lower the solubility of the solute by addition of a
reagent.

Solubility of proteins depend on distribution of

hydrophilic and hydrophobic residues on protein
surface.

Proteins that have high hydrophobic content on the

surface have low solubility in an aqueous solvent.
INTERACTIONS BETWEEN PROTEINS
THAT LEAD TO PRECIPITATION
Electrostatic interactions
 Operate at long range.
 between like molecules are repulsive rather than attractive.
 When surface charge distribution is non-random, it leads to
electrostatic attraction, and finally to association of protein
molecules which causes precipitation.
Hydrophobic interactions
 Main mode of protein- protein interaction.
Van der Waals forces
 Operates at very short range.
 it may prevent molecules coming apart, but probably isn't
important in their coming together.
DRIVING FORCE OF PROTEIN
ASSOCIATION
Non-polar residues at protein surface are shielded by
water molecules.

 When two non-polar protein surfaces come together,

the water molecules are expelled and freed, which
increases their entropy.

This increase in the entropy of water molecules

is the main driving force for protein association
METHODS OF PROTEIN
PRECIPITATION
Reversible
 Salting-out
 Isoelectric precipitation

Irreversible
 Solvent precipitation
 Organic polymers
REVERSIBLE METHODS
SALTING-OUT METHOD
 High concentration of salts such as sodium sulphate,
ammonium sulphate used.
Principle:
 Removal of 'bound' water molecules from hydrophobic
surfaces of the protein, so that they associate by
hydrophobic interaction, which is known to be stronger
at high salt.
 Solubility in (NH4)2SO4 decreases at increasing
temperature; this is used in crystallizing proteins.
Example:

Rabbit muscle glyceraldehyde-3-phosphate

dehydrogenase, precipitation is achieved by adjusting
the pH at constant (NH4)2SO4 concentration.
PROPERTIES OF (NH4)2SO4 MAKING IT MOST
SUITABLE FOR SALTING-OUT METHOD
(NH4)2SO4 is the salt usually used for salting out, because
of its:-

 high solubility (about 3.6 M)

high ionic strength
Its solubility changes little with temperature.
Cheap
Do not react with protein solution.
Good stirring and slow addition should be maintained
while adding (NH4)2SO4, to prevent local high
concentrations of the salt.
The two other purposes of (NH4)2SO4 precipitation
are:-
 Concentration - to diminish the volume in which the
protein is dissolved, for instance after column
chromatography .

 Storage- proteins frequently are particularly stable as

(NH4)2SO4 precipitates.
ISOELECTRIC PRECIPITATION
Isoelectric Point (pI) is the pH of a solution at which the net
primary charge of a protein becomes zero.

pI of most proteins is in the pH range of 4-6.

Mineral acids, such as HCl and H2SO4 are used as precipitants.

Low pH maintained

Net charge goes to zero and all proteins at same pI associate

and precipitate out.

Can be redissolved at another pH and turned to active.

DISADVANTAGE
The greatest disadvantage oF isoelectric point
precipitation is the denaturation caused by the
mineral acids.

For this reason isoelectric point precipitation is most

often used to precipitate contaminant proteins, rather
than the target protein.
IRREVERSIBLE METHOD
SOLVENT PRECIPITATION

 When large amounts of a water-miscible solvent such as

ethanol or acetone are added to a protein solution, proteins
precipitate out.

 Ethanol associates with water much more strongly than do

proteins, so that its real effect is to dehydrate protein
surfaces, which then associate by van der Waals forces.

 Low temperature should be maintained to prevent

denaturation.
PRECIPITATION USING PEG
PEG (polyethylene glycol ) is used at low
concentrations, 5-15% .
Competes with the protein for water.
Less likely to inactivate protein.
Does not require low temperatures.
It tends to give an oily precipitate.
OTHER PRECIPITANTS
Butanol
DOC (deoxycholate)
TCA (trichloro acetic acid)
Methanol/Chloroform
Dextran/PEG, PEG/Salt
Reactors for protein precipitation
Batch reactors
 simplest type of precipitation reactor.

 The precipitating agent is slowly added to the protein solution

under mixing, and the protein aggregates out.

Tubular reactors
 Here, feed protein solution and the precipitating reagent are
contacted in a zone of efficient mixing then fed into long tubes
where precipitation takes place.

 do not require moving mechanical parts and is inexpensive

Continuous stirred tank reactors (CSTR)

run at steady state with a continuous flow of reactants and

products in a well-mixed tank.

 Fresh protein feed contacts slurry that already contains

precipitate particles and the precipitation reagents.