Protein purification
Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity, and biological activity.
Understanding the different protein purification methods and optimizing the downstream processing is critical to minimize production costs while maintaining the quality of acceptable standards of homogeneity.
Several preparative purification steps are often deployed to remove bi-products, such as host cell proteins, which pose a potential threat to the patient's health.
On the other hand, a high yield with low purification levels leaves many contaminants (proteins other than the one interest) which interfere with research purposes.
Depending on how fragile the protein is and how stable the cells are, one could, for instance, use one of the following methods: i) repeated freezing and thawing, ii) sonication, iii) homogenization by high pressure (French press), iv) homogenization by grinding (bead mill), and v) permeabilization by detergents (e.g. Triton X-100) and/or enzymes (e.g.
If the protein of interest is sensitive to proteolysis, it is recommended to proceed quickly, and to keep the extract cooled, to slow down the digestion.
Sometimes it is also necessary to add DNAse in order to reduce the viscosity of the cell lysate caused by a high DNA content.
The rate of centrifugation is determined by the angular acceleration applied to the sample, typically measured in comparison to the g-force.
However, when the proteins are moving through a sucrose gradient, they encounter liquid of increasing density and viscosity.
The use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein.
First, proteins may be purified according to their isoelectric points by running them through a pH-graded gel or an ion exchange column.
The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials.
[7] This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitated protein.
Subsequently, ammonium sulfate can be removed using dialysis (separating proteins from small molecules through a semipermeable membrane).
A detergent such as sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein's native conformation during complete purification.
Consequentially, proteins of a certain range in size will require a variable volume of eluent (solvent) before being collected at the other end of the column of gel.
One chromatography technique based on molecular properties is usually not sufficient in obtaining a protein of high purity.
[1] In addition to size, ion exchange chromatography separates compounds according to the nature and degree of their ionic charge.
Upon injection of the sample, solute molecules will exchange with the buffer ions as each competes for the binding sites on the resin.
Ion exchange chromatography is a very powerful tool for use in protein purification and is frequently used in both analytical and preparative separations.
By making use of a pH-gradient, that can for example be induced by ampholytes, this technique allows to separate protein isoforms up to a resolution of < 0.02 delta-pI.
[8] Additionally, the environment used typically employs less harsh denaturing conditions than other chromatography techniques, thus helping to preserve the protein of interest in its native and functional state.
[9] Affinity Chromatography is another powerful separation technique that is highly selective for the protein of interest based upon molecular conformation, which frequently utilizes application specific resins.
This "lock and key" fit between the ligand and its target compound makes it highly specific, frequently generating a single peak, while all else in the sample is unretained.
Detergent-solubilized proteins can be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin.
Immunoaffinity chromatography uses the specific binding of an antibody-antigen to selectively purify the target protein.
The procedure involves immobilizing a protein to a solid substrate (e.g. a porous bead or a membrane), which then selectively binds the target, while everything else flows through.
Immunoprecipitation is capable of generating an extremely specific interaction which usually results in binding only the desired protein.
The latter can be determined by the Bradford total protein assay or by absorbance of light at 280 nm, however some reagents used during the purification process may interfere with the quantification.
A non-denaturing electrophoretic procedure for isolating bioactive metalloproteins in complex protein mixtures is preparative native PAGE.
