Purification of proteins

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Aug 10, 2008 Views: 692

Purification of proteins

A protein must be purified before its structure and the mechanism of its action can be studied. However, because proteins vary in size, charge, and water solubility, no single method can be used to isolate all proteins.
To isolate one particular protein from the estimated 10,000 different proteins in a cell is a daunting task that requires methods both for separating proteins and for detecting the presence of specific proteins.
Any molecule, whether protein, carbohydrate, or nucleic acid, can be separated from other molecules based on large differences in some physical characteristic.
Although the sequence of amino acids in a protein uniquely determines its function, the most useful physical characteristic for separation of proteins is size, defined as either length or mass.
In this section, we briefly outline different techniques for separating proteins based on their size and other properties.
These techniques also apply to the separation of nucleic acids and other biomolecules. We then consider general methods for detecting, or assaying, specific proteins, including the use of radioactive compounds for tracking biological activity.
Finally, we discuss several techniques for characterizing a protein's mass, sequence, and three-dimensional structure.
Liquid Chromatography Resolves Proteins by Mass, Charge, or Binding Affinity
- Liquid chromatography, a third commonly used technique to separate mixtures of proteins, nucleic acids, and other molecules, is based on the principle that molecules dissolved in a solution will interact (bind and dissociate) with a solid surface.
- If the solution is allowed to flow across the surface, then molecules that interact frequently with the surface will spend more time bound to the surface and thus move more slowly than molecules that interact infrequently with the surface.
- Liquid chromatography is performed in a column packed tightly with spherical beads. The nature of these beads determines whether separation of proteins depends on differences in mass, charge, or binding affinity.
- Gel Filtration Chromatography
- Proteins that differ in mass can be separated by gel filtration chromatography. In this technique, the column is composed of porous beads made from polyacrylamide, dextran (a bacterial polysaccharide), or agarose (a seaweed derivative).
- Proteins flow around the spherical beads in gel filtration chromatography. However, the surface of the beads is punctured by large holes, and proteins will spend some time within these holes.
- Because smaller proteins can penetrate into the beads more easily than larger proteins, they travel through a gel filtration column more slowly than larger proteins . (In contrast, proteins migrate through the pores in an electrophoretic gel; thus smaller proteins move faster than larger ones.)
- The total volume of liquid required to elute a protein from the column depends on its mass: the smaller the mass, the greater the elution volume. By use of proteins of known mass, the elution volume can be used to estimate the mass of a protein in a mixture.
- Ion-Exchange Chromatography
- In a second type of liquid chromatography, called ion-exchange chromatography, proteins are separated based on differences in their charge.
- This technique makes use of specially modified beads whose surfaces are covered by amino groups or carboxyl groups and thus carry either a positive charge (NH₃⁺) or a negative charge (COO⁻) at neutral pH.
- The proteins in a mixture carry various net charges at any given pH. When a solution of a protein mixture flows through a column of positively charged beads, only proteins with a net negative charge (acidic proteins) adhere to the beads; neutral and basic proteins flow unimpeded through the column .
- The acidic proteins are then eluted selectively by passing a gradient of increasing concentrations of salt through the column.
- At low salt concentrations, protein molecules and beads are attracted by their opposite charges. At higher salt concentrations, negative salt ions bind to the positively charged beads, displacing the negatively charged proteins.
- In a gradient of increasing salt concentration, weakly charged proteins are eluted first and highly charged proteins are eluted last. Similarly, a negatively charged column can be used to retain and fractionate positively charged (basic) proteins.
- Affinity Chromatography
- A third form of chromatography, called affinity chromatography, relies on the ability of a protein to bind specifically to another molecule.
- Columns are packed with beads to which are covalently attached ligand molecules that bind to the protein of interest. Ligands can be enzyme substrates or other small molecules that bind to specific proteins. In a widely used form of this technique, antibody-affinity chromatography, the attached ligand is an antibody specific for the desired protein .
- An affinity column will retain only the proteins that bind the ligand attached to the beads; the remaining proteins, regardless of their charge or mass, will pass through the column without binding to it.
- The proteins bound to the affinity column then are eluted by adding an excess of ligand or by changing the salt concentration or pH. Obviously, the ability of this technique to separate particular proteins depends on the selection of appropriate ligands.