advances-in-protein-chemistry

An overview of various purification techniques is given in the following sections. The description of techniques used in protein
purification in the upcoming sections has been kept short and some minute details have been skipped in order to keep the chapter concise.
Fractionation of proteins in solution
Almost invariably, the target protein is obtained in aqueous solution together with several undesirable proteins impurities. The
fractionation techniques may provide a way to remove at least some of the impurities while reducing the bulk of the sample. Although
fractionation does only lead to a very partial purification, it generally precedes the high resolution techniques. In this way, a crude sample
is partially purified and its volume is reduced to make it suitable to be processed further by more sophisticated methods. Depending upon
the properties of the target protein, one of the following methods may be used for fractionation.
Fractionation by precipitation: This method utilizes the property of the differential solubility of proteins under different conditions
as well as conditions which allow aggregation of proteins. The precipitation may be enforced by altering the buffer composition. The
precipitated protein may be separated from other simply by low speed centrifugation. One of the following methods may be used to
achieve this objective:
a. Salt fractionation: High ionic strength promotes protein precipitations. With increasing ionic strength, proteins begin to interact
via hydrophobic patches on their surface, as proteins and salt compete for the residual water. This leads to formation of aggregates and
the precipitation of proteins. The phenomenon is termed as “Salting out” of proteins. Different proteins would precipitate at different salt
concentrations. Ammonium sulfate is used as the salt of choice, since it preserves protein activity and promotes precipitation at lower
concentrations than other salts. Other salts may give low ionic strength (e.g. NaCl, KCl), have solubility problems (e.g. Na 2
SO 4
) or may
cause damage to protein structure. Increasing amount of ammonium sulfate is added to the extract, with intermittent centrifugation
steps. These stepwise precipitations are referred to as ammonium sulfate “cuts”. The presence of target protein is checked in these cuts
and an appropriate cut is determined where the major portion of target protein is concentrated. Pre-calculated tables are available to
find out the amounts of solid ammonium sulfate to be added to a known volume in order to obtain the desired percentage saturation.
b. Isoelectric precipitation: Apart from ionic strength, the solubility of proteins also depends on pH. As the pH approaches the
pI of a given protein the net charge on that protein gets closer to zero, and weak electrostatic attractive forces lead to aggregation
and precipitation. Lowering ionic strength, or addition of solvent, promotes these interactions. Although, solubility is minimal at the
isoelectric point, the increase in ionic strength increases the solubility even at the isoelectric point (This effect is called “salting in” of
proteins).
c. Solvent precipitation: The mechanism of solvent fractionation is reduction of availability of water for protein solvation (i.e. a
reduced water activity) which causes the precipitation of the protein. It tends to be more effective at pI which suggests that the mechanism
may be similar to that in isoelectric precipitation. The precipitation may be achieved by adding water-soluble organic solvents such as
ethanol or acetone. The concentration of solvent required depends on the type of protein and usually ranges between 5 to 60% (v/v). Since
the denaturing effect of solvents increases with temperature, solvent fractionation is generally carried out around 0°C. The method has
been extensively used in the past but nowadays only done in few specific cases. The main disadvantage of the method is the denaturation
of proteins due to non-polar solvents. However, if the protein of interest is known to be stable in these conditions, the method may be
worthwhile to use since it will remove many of unwanted proteins that are sensitive to solvents.
d. Selective denaturation: Contaminant proteins in isolated cases can be selectively denatured by heat, extremes of pH, and organic
solvents. These treatments may be carried out if the protein of interest is stable towards one or more of these conditions. However, the
treatment may result in chemical modification of the desired protein.
It is important to note that whole of the protein of interest may not come in a single fraction. Many times, some of the protein has to
be sacrificed in order to get a better elimination of impurities.
Ultra filtration: Dialysis, that employs a semi permeable membrane made from cellulose, is frequently used for desalting purposes.
The protein is retained in the dialysis bag while the salt moves out. If the membrane has larger pores that also allow small proteins to move
out while retaining the larger ones, such membranes can be used for protein fractionation. Since dialysis speed is a function of molecular
mass, the movement of proteins through dialysis tubing would be insignificant. Ultrafiltration, on the other hand, uses such membranes
and employs pressure to force the sample through. Membranes ranging from 10kD to 100kD molecular weight cutoffs are commercially
available. The method is not very efficient due to significant loss of sample, but may be useful in certain specific cases.
In order to increase the processing potential and the specificity of membrane filtration, affinity ultrafiltration concept was introduced.
Affinity ultrafiltration follows the principle that
a. When the target protein and impurities are free in a solution pass via the ultrafiltration membrane.
b. Whereas, when a macro-ligand is bound to the target protein, the resultant is a membrane-retained complex.
After the target protein binds to the macroligand, the impurities are washed away. Dissociation of the target protein from macroligand
can be performed by providing suitable conditions.
Solid Phase Extraction (SPE)
Solid Phase Extraction (SPE) is method that uses a solid phase and a liquid phase to isolate one type of analyte from others based
on physic-chemical properties. It is generally used to concentrate and/or clean up a sample before using a chromatographic or other
analytical method. Solid-phase extractions use the same type of stationary phases as are used in liquid chromatography (discussed in
later sections).
SPE uses the affinity of solutes dissolved or suspended in a liquid phase (the mobile phase) for a solid phase (the stationary phase)
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