Recombiant Protein Purification

What is Protein Purification?

Deffinition of Protein Purification

The expression of recombinant proteins, especially using bacterial vectors and hosts, is a mature technology. The problem is how to isolate it in an active form.

Purification of recombinant protein is an important technology in biological research. To study the particular function and structure of a protein, researchers must isolate and purify the recombinant protein from the organism. The protein purification method mainly uses the similarity and difference between different recombinant proteins. Non-proteinaceous materials can be removed based on the similarity between proteins, and the target recombinant protein then can be isolated and purified based on the differences between proteins.

Protein tags are a useful and convenient tool for improving solubility of recombinant proteins, streamlining protein purification, and allowing an easy way to track proteins during protein expression and purification. In addition, protein tags are a useful tool for tracking proteins and processes directly in live cells using microscopy or indirectly using Western blot, immunoprecipitation, or immunostaining.

Protein Purification Principles and Practice

The Principle of Protein Purification

Different recombinant proteins have different amino acid sequences and spatial structures, resulting in differences in their physical, chemical, and biological properties. A reasonable protein purification scheme can be designed based on the differences in the properties of the desired protein to be separated from other proteins and lysates.

Most purification protocols require more than one step to achieve desired level of purity. Each step in the process will cause some loss of product, a yield of 80% in each step is assumed, and therefore, it is advisable to have as few purification steps as possible. Choice of a starting material is key to the design of a purification process.

With background information, assays and sample procedures in place the three phase purification strategies can be considered. The purification has three phases of capture, intermediate purification and polishing, each with specific objective. In capture phase the objectives are to isolate, concentrate and stabilize the target procedure.

Protein Purification Practice

The final purification process ideally consists of sample preparation, including extraction and clarification when required followed by above described three phases of purification. The number of steps will always depend on purity required and intended use of protein.

An analytical purification generally utilizes three properties to separate proteins. First, proteins- may be purified according to their isolectric points by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) analysis. Proteins are often purified by using 2D-PAGE and are then analyzed by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific purposes and the detection limits for protein are nowadays very low and Nano gram amounts of protein are sufficient for their analysis.

How to Apply Purification Principles

Selection and Combination of Purification Techniques:
The aim of this combination is to evolve a fastest route to a product of required purity. For any chromatographic separation each different technique will offer a different performance with respect to recovery, resolution, speed and capacity. A technique can be optimized to focus on one of these parameters; for example, resolution to achieve the best between two parameters such as speed and capacity.

Resolution is achieved by the selectivity of the technique and efficiency of the chromatographic matrix to produce narrow peaks. In general, resolution is most difficult to achieve in final phases of purification where target protein and impurities have very similar properties.

Purification of a Tagged Protein:
Adding a tag to the protein gives the protein a binding affinity it would not otherwise have. Usually the recombinant protein is the only protein in the mixture with this affinity, which aids in separation. The most common tag is the Histidine-tag (His-tag) that has affinity towards nickel or cobalt ions. Thus by immobilizing nickel or cobalt ions on a resin, an affinity support that specifically binds to histidine tagged proteins can be created.

Evaluating Purification Yield:
The most general method to monitor the purification process is by running a SDS PAGE, of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar molecular weight. In order to evaluate the process of multistep purification, the amounts of the specific protein have to be compared to the amount of total protein.

References

Wingfield PT (2015) Overview of the purification of recombinant proteins. Curr Protoc Protein Sci 80: 6.1.1-6.1.35.
Ferrer-Miralles N, et al. (2015) General introduction: Recombinant protein production and purification of insoluble proteins. Methods in molecular biology (Clifton, N.J.) 1258: 1-24.
Pina AS, et al. (2014) Affinity tags in protein purification and peptide enrichment: An overview. Methods in molecular biology (Clifton, N.J.) 1129: 147-168.

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