Protein Purification

– Protein Identification

Protein purification begins with the need to identify the protein we want to purify! There are several methods that can be used to rapidly identify the protein:

  • Enzyme Assay (by catalytic activity) – with certain enzymes we can use colorimetry to detect a product as a reaction progresses. The higher the more enzyme present, the faster the colour or light absorbance will change. An example is testing for Alcohol Dehydrogenase, which will lead to a change in the levels of NADH and NAD+ as Ethanol is converted to Ethanal. This change can be detected by colorimetry at ~340nm.
  • SDS-PAGE Electrophoresis (by size) – this method seperates protein chains by size by electrophoresis. This method denatures the proteins.
    The sample is run at the same time as a molecular mass marker sample, containing proteins of known mass. The marker sample will provide a scale for the mass of your sample. Once you’ve run the gel you will be able to plot the results as above, draw a best fit line and read off the Molecular Mass of your sample protein.
  • Immuno-Assay (by specific antibodies) – Antibodies that fit specifically to the protein you are looking for are added to the sample. When these bind to the target protein they will instigate a colour change or some other noticable change. The presence and concentration of the target protein can be assessed by the extent of the changes – if it was a colour change then the darker the colour goes, the more target enzyme must be present.
  • Western Blotting – A combination of electrophoresis and immuno-assay. The immuno-assay technique is run by electrophoresis. This will be useful if your sample contains several different proteins and you need to identify the target protein. The band of colour (or change) will show you the correct protein, and then you simply need to calculate the approximate molecular mass using the molecular mass markers.

– Protein Purification

I’ve broken the purification methods here into 8 different headers, each a physical-chemical property or biological activity.

  1. Stability (Heat). Some proteins are more heat tolerant than others and can survive heating while others denature. If your target protein is heat stable at above 60C and your contaminants are not, then simply heating your mixture to 60C for 30 minutes will denature most of the contaminents. This will leave you with a much higher concentration of your target protein in your mixture.
  2. Solubility (Seperate by pI). Proteins are least soluble at the pH equal to their isoelectronic point. When helped by the addition of salts to the solution this can lead to their precipitation. As the salt concentration increases, different proteins will precipitate.
  3. Size. Proteins can be seperated by Gel Permeation Chromatography (Gel filtration). The proteins are run through a buffered, porous, cross linked resin. While small molecules are able to fit into the pores in the resin, the larger proteins cannot and so travel ahead, with the small molecules lagging behind.
    This is due to a larger volume of buffer available to the smaller molecules, meaning more buffer must pass down the column for them to elute, compared to the relatively smaller volume of buffer required to elute the larger, excluded proteins.
  4. Density (Centrifuge). By centrifuging the sample in a test tube containing a sucrose density gradient, the centrifugal forces will force the proteins down the tube until they reach a concentration where the density of the sucrose solution is the same as their own. This level is known as it’s isopycnic level.
  5. Charge. There are several different methods of purification by charge:
    1. Gel Electrophoresis – Based on movement of a protein through a cross linked gel called polyacrylamide. This would occur at a pH where the protein has a charge (not at it’s pI). The size of the pores can be altered by changing the concentration of cross linking reagent, and the speed at which a protein travels is equal to its charge:mass ratio. (This method does not tell us anything about the protein’s molecular weight).
    2. SDS PAGE – This cannot really be used for purification because SDS detergent (Sodium Dodecylsulphate) is used which denatures the protein. It unfolds the protein and surrounds it with -ve charge sulphate groups which means all the proteins have a uniform charge:mass ratio. SDS has a 12 carbon hydrocarbon chain, and then a hydrophilic sulphate group. The Sulphate groups surrounding the protein form a miscelle.
      The sample is now allowed to run on a gel, from -ve to +ve, and as they all have the same mass to charge ratio, their rate is determined only by their size. The smaller protein molecules move faster and the larger molecules move slower through the gel.
    3. Isoelectric Focusing РVery similar to (1) above, but instead of an electric charge, there is a pH gradient along which the proteins can move until they are at a point where they  have no net charge (at their pI).
    4. Ion Exchange Chromatography. Essentially, both columns and proteins become charged at different pH’s, and by altering the pH we can hold on to some proteins while others are eluted.
      Diethylaminoethyl-Cellulose (DEAE-Cellulose) has a +ve charge below pH 9.5 wheras CarboxyMethyl-Cellulose (CM-Cellulose) has a -ve charge above pH 3.0. Therefore:
      – Proteins with a +ve charge at pH7 will bind to a column of CM-Cellulose, while
      – Proteins with a -ve charge at pH7 will bind to a column of DEAE-Cellulose.
      We can then alter the pH of the solution to release certain proteins or to pick up others. Another way of dispersing ionic interactions between the column and proteins is to increase the salt concentration.
  6. Hydrophobicity. Proteins nearly always feature hydrophobic areas or side chains and these allow the proteins to bind to resins with hydrophobic groups attached. This means the proteins can be eluted with a gradient of buffer (eg an organic solvent such as ethanol). The proteins forming the strongest interactions with the resin column will require higher concentrations of ethanol to elute.
  7. Biological Function. If a protein has a high affinity for a substrate (eg. ADH has a high affinity for NAD+) then we can use affinity chromatography. If we immobilise the substrate (eg. NAD+) then the protein will bind to that substrate, immobilising itself – allowing other proteins to run free of the column. By releasing free NAD+ throught he column the substrate will gradually release the immobilised NAD+ in favour of the free NAD+ and run free of the column.
    This method can purify a protein in one step, and works best if the protein has a high affinity for the bound ligand.
  8. Fusion Proteins. This involves the addition of a gene to a protein that essentially ‘tags’ the protein. An example would be a tag containing histidine residues, which would bind to metal ions in the column.
    Here, the imidazole rings on the histidine residues stick to the immobilised metai ions allowing other proteins to elute the column. Then, like the method above, add free imidazole to release the fusion proteins and then use a protease to cut the tag away. Run the column again and only the tags will bind, allowing the protein of interest to run free.

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