Steps to Peptide/Protein Purification:
Peptides and proteins take place naturally as parts of tissues or cells. First step in protein purification procedure is generally cell disruption. Objective of this step is to release protein content of the cells/tissues. Cell disruption is performed using blender or pestle and mortar, and at times by repeated cycles of icing and thawing. After cell disruption, crude extract is then centrifuged to divide cellular pellets or debris from clear solution that contains mixture of proteins. This clear supernatant is consequently subjected to other purification protocols until protein of interest is isolated.
Purification Technique on the Basis of Solubility:
Early fractionation steps in protein/peptide purification use differences in protein/peptide solubility. At high ionic strengths, solubilities of polypeptides decrease with increasing salts concentration. This event is known salting out and it arises due to competition between added salt ions and dissolved polypeptides for molecules of solvation. Many solute ions are so solvated that polypeptides aren't adequately solvated but forced to precipitate out of solution. Precipitation techniques are popular first steps in protein purification as they can be performed in large batch scale.
Addition of certain salts in correct amount can selectively precipitate some polypeptides, whereas others remain in solution. Ammonium Sulphate is the most normally utilized reagent for salting out proteins. Once precipitated, proteins are then removed or concentrated by filtration or centrifugation. Organic solvents like acetone and ethanol are also good precipitating agents. On the other hand, salting in refers to increase in protein solubility at low ionic strength with increase in salt concentration.
Purification Technique on the Basis of Molecular Size:
i) Dialysis:
Dialysis is a separation procedure in which macromolecules like polypeptides are separated from smaller molecules of solvent, salts, minerals and other metabolites. This is done with the aid of semi permeable membrane such as cellophane (made of cellulose acetate), taking advantage of variation in molecular size. The solution containing the protein mixture is sealed in a dialysis bag (see figure 1below). When the sealed bag is immersed in a much larger volume of buffered solution and allowed some time to equilibrate, the membrane barrier allows the flow of the small molecules out of the bag but not the proteins. The flow of these molecules is via osmosis and it can be enhanced by stirring. By continuously putting the bag in fresh buffer solution, it is possible to get rid of the smaller salts ions.
ii) Ultracentrifugation:
This is another separation process in which proteins are purified on the basis of molecular masses or sizes. When the solution of macromolecules is subjected to the ultracentrifugal force (the force greater than 4 x 105 times that of gravity), proteins accelerate quickly to the constant velocity of sedimentation. This is stated as sedimentation constant, S. S is rate per unit of centrifugal force and is provided by equation below:
S = v/w2r
Where v is velocity of protein, W is angular velocity of centrifuge and r is radius of rotation that is distance from center of tube in which protein mixture is put to centre of rotation.
Each protein sediment's with the characteristic sedimentation coefficient. Unit of s is Svedberg (S) named after Swedish biochemist who first developed ultracentrifuge in 1923. (1S= 10-13).
Ultracentrifugation has been utilized in determination of proteins' molecular weights by just taking benefit of given equation:
S = v/W2r=(M1-v‾ ρ)/Nf
M= molecular mass in daltons
v‾ = partial specific volume in ml/gram
ρ (rho)= density (g/ml) of the solvent.
f = frictional coefficient
In order to achieve enhanced resolution of the macromolecules, improved process of sedimentation which involves conducting the process in density gradient solution of inert substances such as sucrose or CsCl has been developed. This version of ultracentrifugation is called density gradient ultracentrifugation.
iii) Gel Filtration:
This separation method is also known as molecular exclusion, size exclusion and molecular size chromatography. It divides molecules of proteins/peptides according to the sizes and shape. It is column chromatography and like all chromatographic methods, it is composed of stationary and mobile phases. Protein samples dissolved in liquid solvent make up mobile phase whereas stationary phase comprises of small insoluble matrix or beads of hydrated, sponge-like material called as gel. Gel has pores or cavities of the particular size. If the solution of proteins of different sizes is permitted to pass through this gelcontaining column, gel acts as the molecular sieve allowing their elution on the basis of size. Small proteins penetrate pores of gel and contain larger solvent volume by which to journey down column (than larger proteins) and so are eluted last. Larger proteins transverse the column more rapidly as they are not trapped in gel cavities. Thus, larger polypeptides are first to be eluted from column.
Kinds of gel materials:
Different kinds of materials have been utilized as gel materials. Common ones comprise:
1. Dextran (high molecular mass polymer of glucose made by bacterium Leuconostoc mesenteroides). Dextran is sold under trade name Sephadex.
2. Agarose (linear polymer of alternating D-glucose and 3.6-anhydro-L-galactose attained from algae. It is commercially available as Sephapose and Bio-Gel A.
3. Polyacrylamide (commercially available as Bio-Gel P). Polyacrylamide gels are largely used for separation of proteins.
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