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Protein Purification

Traditionally protein purification methods have often resulted in the separation of protein complexes for the sake of "purity" or have inadvertently destroyed complexes as a result of the chemical or physical basis of the separation procedure.

Sometimes the complex can be reconstituted by combining individually purified proteins, but in many cases activity cannot be recovered. In addition it is often difficult to identify the binding partners once they are physically separated.

 

Protein Purification Methods

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Complex protein mixtures were fractionated mainly by adsorption and precipitation methods. These methods are still used today as preliminary steps for initial fractionation or for concentration of sample solutions.

Preparative electrophoretic and chromatographic techniques developed during the 1950's and 1960's made possible rational purification protocols and laid the foundation for the situation. The following are the methods used for the protein purification.
  • Precipitation

Precipitation of a protein in an extract may be achieved by adding salts, organic solvents or organic polymers or by varying the pH or temperature of the solution.

  • Electrophoresis

Electrophoresis in free solution or in macro porous gels such as 1-2% agarose separates proteins mainly according to their net electric charge. Electrophoresis in gels such as poly-acrylamide separates mainly according to the molecular size of the proteins.

  • Chromatography

Separation by chromatography depends on the differential partition of proteins between a stationary phase and a mobile phase. Normally, the stationary phase is packed into a vertical column and the buffer is pumped through this column. An alternative is to stir the protein solution with the adsorbent. Pour the slurry onto the appropriate filter and make the washings and desorption on the filter.

Column chromatography has proved to be an extremely efficient technique for the separation of proteins in biological extracts.

  • Expanded bed adsorption

In a typical fluidized bed there is a total mixing of particles and sample in the reactor leading to incomplete adsorption of the target molecules unless the feed stock is recycled. Expanded bed adsorption is a special case of fluidized bed adsorption and is primarily applied in a pilot or production scale environment.

  • Membrane adsorption

In membranes, most pores allow convective flow, and the mass transport resistance is therefore minimized to film diffusion at the membrane matrix surface. The result is a more efficient adsorption-desorption cycle of target solutes, allowing considerably higher flow rates and thus considerably shorter separation times.

Protein Purification Tags

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Protein purification tags are protein sequences that possess high-affinity binding properties for particular molecules, and the tag allows the target protein to bind to a solid support usually in the form of column matrix, to which very few other proteins are able to bind.
  • HIS-tag

The simplest of all protein purification tags the HIS (histidine) tag is normally composed of six histidine residues. The DNA encoding these residues is cloned into the target gene such that the produced protein contains at some point in its polypeptide sequence six consecutive histidine residues.

Cloning is often performed such that the tag is located at either the extreme amino or extreme carboxy terminal end of the protein, where it is less likely to impair protein function.
The tag may, however also be placed in the middle of a protein if a central region is already known to be non essential for function.

  • GST-Tag

A GST ( glutatione S - transferase) tag appends glutathione S-transferase to the protein of interest. Typical GST tags are on the order of 220 amino acids and can be used for protein purification by binding of the tagged protein to a solid support containing immobilized glutathione. Antibodies are available for immuno-histochemical analysis of proteins containing GST-tags.

Each and every preparation should be continually recorded in a purification table. In combination with results from gel electrophoresis, for example this will serve as a guide for judging the re productivity and outcome of each preparation.

Protein Purification Table

Material
Volume(ml)
Protein(mg/ml) Total protein(mg) Activity(U)
Total activity(U)
Specific activity(U/mg)
Yield(%)
Purification factor (fold)
Extract 500 14 7000 7 3500 0.5 100 1
First purification step 50 10 500 60 3000 6 85 12

Downstream Protein Purification

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  1. The basic aim when designing a downstream purification protocol is to keep the number of unit operations to the bare minimum so that an essentially "pure" product is obtained with the highest possible recovery and at a minimum cost.
  2. In many applications HIC (Hydrophobic interaction chromatography) is particularly suitable to use after techniques that leave the sample in a high salt concentration as the sample can often be applied directly.
  3. Used as a first step in a downstream purification processes, HIC can also serve as an effective step for concentrating dilute samples.
  4. In contrast the only conditioning that is necessary when using HIC is to add sufficient salt to the sample to promote the binding of the soluble to the adsorbent.
  5. In instances where the biological extract has been precipitated by high concentrations of ammonium sulfate, HIC fits in naturally as the first step of a downstream purification processes.
  6. Purification of proteins using affinity chromatography allows the possibility of obtaining several fold purification with high recovery in fewer steps.
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